Methods of use in support of planned supplement and food ingredient products for diagnosis and treatment of anemia

ABSTRACT

Compositions including a fatty acid, such as an odd chain fatty acid or a very long even chain fatty acid, and salts and derivatives thereof, and methods of use in support of planned supplement and food ingredient products for diagnosis and treatment of anemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/267,570, which is a divisional of U.S. patent application Ser. No.15/393,771, filed on Dec. 29, 2016, entitled “Composition and Methodsfor Diagnosis and Treatment of Anemia,” the content of which is fullyincorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

The United States Government has ownership rights in this invention,pursuant to passing of title to a Subject Invention under Federal GrantN00014-15-1-2131 (National Marine Mammal Foundation). Licensinginquiries may be directed to Office of Research and TechnicalApplications, Space and Naval Warfare Systems Center, Pacific, Code72120, San Diego, Calif., 92152; telephone (619) 553-5118; email:ssc_pac_t2@navy.mil, referencing NC 111500.

FIELD OF THE INVENTION

Compositions including fatty acids, and salts and derivatives thereof,and methods for treatment or prophylaxis of anemia are provided,including compositions and methods for treating anemic conditions suchas hemolytic anemia and anemia of chronic disease, and other relatedconditions.

BACKGROUND OF THE INVENTION

Anemia is a blood condition indicated by detection of low hemoglobin,low hematocrit, low packed cell volume, and/or the oxygen-carryingcapacity in the blood. Anemia is the most common blood disorder in theUnited States, affecting over 3 million people a year. Anemia isindependently associated with poorer health outcomes in people withchronic diseases, including an increased risk of developing cardiacdisease and an increased risk of mortality in end stage renal diseasepatients. The annual cost of underlying diseases for people withconcurrent anemia of chronic disease (ACD) is double that of people withchronic diseases without ACD. Thus, anemic diseases are implicated in awidespread public health problem. Types of anemia include inheritedforms, such as thalassemia and sickle cell anemia, those in which redblood cell (RBC) production is low, those in which red blood cells areabnormal in shape or size, and those in which red blood cells undergoelevated rates of cell death. Symptoms of anemia include fatigue,weakness, pale or yellowish skin, irregular heartbeats, shortness ofbreath, dizziness or lightheadedness, chest pain, cold hands and feet,and headache. Anemia can further be associated with the risk ofdeveloping heart problems, severe fatigue, pregnancy complications, anddeath. Anemia can impair cognitive development and reduce physical workcapacity. Risk for anemic conditions is associated with a diet lackingin certain vitamins, intestinal disorders, menstruation, pregnancy,family history of anemia, blood diseases, autoimmune disorders,alcoholism, exposure to toxic chemicals, chronic inflammation, the useof some medications, and poor health arising from chronic conditions.

SUMMARY OF THE INVENTION

Compositions and methods for treatment or prophylaxis of anemia areprovided. These compositions comprise one or more fatty acids,derivatives of fatty acids, or salts thereof, which may be administeredin combination with other medicaments or as part of various treatmentregimens as described herein. The provided compositions are effectivefor modulating markers associated with anemia. Methods are provided foradministering the compositions.

Accordingly, in a generally applicable first aspect (i.e., independentlycombinable with any of the aspects or embodiments identified herein), apharmaceutical composition is provided, comprising: one or more fattyacids, or pharmaceutically acceptable salts thereof, wherein the one ormore fatty acids are selected from the group consisting of odd chainfatty acids and very long even chain fatty acids; and a pharmaceuticallyacceptable carrier.

In an embodiment of the first aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), the one ormore fatty acids is heptadecanoic acid.

In an embodiment of the first aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), thecomposition is substantially free from even chain fatty acids.

In an embodiment of the first aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), the one ormore fatty acids is behenic acid.

In an embodiment of the first aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), thecomposition comprises at least one odd chain fatty acid and at least onevery long even chain fatty acid.

In an embodiment of the first aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), the one ormore fatty acids comprises heptadecanoic acid and behenic acid.

In an embodiment of the first aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), thecomposition is in a unit dosage form.

In an embodiment of the first aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), thepharmaceutical composition is configured for administration of from 1.0mg to 11 mg, per 1 kg of body weight, of the one or more fatty acids orpharmaceutically acceptable salts thereof to a patient.

In an embodiment of the first aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), thepharmaceutical composition is configured for administration once perday.

In an embodiment of the first aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), thepharmaceutical composition comprises from 0.01 mg to 10000 mg of the oneor more fatty acids or pharmaceutically acceptable salts thereof.

In a generally applicable second aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), use isprovided of a pharmaceutical composition of the first aspect or anyembodiment thereof, in the manufacture of a medicament for treatment orprophylaxis of a method is provided for treatment or prophylaxis of ananemic condition, hemolytic anemias (including but not limited tothalassemias, hereditary spherocytosis, hereditary elliptocytosis,glucose-6-phosphate dehydrogenase deficiency, pyruvate kinasedeficiency, immune hemolytic anemia, alloimmune hemolytic anemia,drug-induced hemolytic anemia, mechanical hemolytic anemias, andparoxysmal nocturnal hemoglobinuria), anemia of chronic disease, anemia,aplastic anemias (including but not limited to congenital hypoplasticanemia, Diamond-Blackfan anemia and Fanconi anemia), iron deficiencyanemia, anemias of abnormal RBC size (including but not limited tomegaloblastic anemia and microcytic anemia), vitamin deficiency anemias(including but not limited to pernicious anemia) anemia of RBC mutation(including but not limited to thalassemia, sideroblastic anemia andsickle cell anemia).

In a generally applicable third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), a method isprovided for treatment or prophylaxis of an anemic condition, hemolyticanemias (including but not limited to thalassemias, hereditaryspherocytosis, hereditary elliptocytosis, glucose-6-phosphatedehydrogenase deficiency, pyruvate kinase deficiency, immune hemolyticanemia, alloimmune hemolytic anemia, drug-induced hemolytic anemia,mechanical hemolytic anemias, and paroxysmal nocturnal hemoglobinuria),anemia of chronic disease, anemia, aplastic anemias (including but notlimited to congenital hypoplastic anemia, Diamond-Blackfan anemia andFanconi anemia), iron deficiency anemia, anemias of abnormal RBC size(including but not limited to megaloblastic anemia and microcyticanemia), vitamin deficiency anemias (including but not limited topernicious anemia) anemia of RBC mutation (including but not limited tothalassemia, sideroblastic anemia and sickle cell anemia), comprising:administering to a patient in need thereof, an effective amount of oneor more fatty acids, or pharmaceutically acceptable salts thereof,wherein the one or more fatty acids are selected from the groupconsisting of one or more odd chain fatty acids, one or more very longeven chain fatty acids, and combinations thereof.

In an embodiment of the third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), the one ormore fatty acids or pharmaceutically acceptable salts thereof isprovided as a pharmaceutical composition in a unit dosage formcomprising the one or more fatty acids or pharmaceutically acceptablesalts thereof and a pharmaceutically acceptable carrier.

In an embodiment of the third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), the unitdosage form comprises from 0.01 mg to 10000 mg of the one or more fattyacids or pharmaceutically acceptable salts thereof.

In an embodiment of the third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), the one ormore odd chain fatty acids is heptadecanoic acid.

In an embodiment of the third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), thepharmaceutical composition is substantially free from even chain fattyacids.

In an embodiment of the third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), the one ormore very long even chain fatty acids is behenic acid.

In an embodiment of the third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), thepharmaceutical composition comprises a plurality of different fattyacids.

In an embodiment of the third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), from 1.0 mgto 11 mg of the one or more fatty acids or pharmaceutically acceptablesalts thereof is administered to the patient, per 1 kg of body weight,per day.

In an embodiment of the third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), the one ormore fatty acids or pharmaceutically acceptable salts thereof isadministered to the patient once per day.

In an embodiment of the third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), a serumconcentration or a red blood cell membrane concentration of the one ormore fatty acids is increased by at least about 0.001×10⁻⁴ M above apretreatment value.

In an embodiment of the third aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), thepharmaceutical composition is configured to modulate a marker of anemiaor a symptom of anemia selected from the group consisting of serum orred blood cell membrane very long even chain fatty acid percentage,serum concentration of a very long even chain fatty acid, serum totalvery long even chain fatty acid, serum ferritin, serum iron, transferrinsaturation, red blood cell count, reticulocytes, hematocrit or packedcell volume, red blood cell distribution width, mean corpuscularhemoglobin concentration (MCHC), mean corpuscular volume (MCV), meancorpuscular hemoglobin (MCH), hemoglobin, white cell count, irondeficiency, serum iron, total iron-binding capacity, transferrinsaturation, zinc protoporphyrin, and serum sTfRs.

In a generally applicable fourth aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), a compositionsubstantially as described herein is provided.

In a generally applicable fifth aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), a compositionsubstantially as described herein is provided.

In a generally applicable sixth aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), a usesubstantially as described herein is provided.

DESCRIPTION OF THE DRAWINGS

FIG. 1. provides data on improving hematocrit among individual dolphinswhile on the modified fish diet.

FIGS. 2A-2D provide data for positive associations between serumconcentrations of cholesterol ester C15:0 and phosphatidylcholine C17:0,with hematocrit and hemoglobin, using simple linear regression models indolphins on a modified fish diet.

FIG. 3 provides data for serum total C17:0 (as a % of total fatty acids)and hematocrit (%), with demonstration of a therapeutic C17:0 valuethreshold protective against anemia (hematocrit lower than 40%) using asimple linear regression model in dolphins.

FIGS. 4A-4C provide data for positive associations between serumconcentrations of hexosylceramide forms of very long even chainsaturated fatty acids (C20:0, C22:0 and C24:0) with hematocrit, usingsimple linear regression models in dolphins on a modified fish diet.

FIGS. 5A-5D provide data for positive associations between serumconcentrations of cholesterol ester C15:0 and phosphatidylcholine C17:0with hexosylceramide forms of very long even chain saturated fatty acids(C22:0 and C24:0), using simple linear regression models in dolphins ona modified fish diet.

FIG. 6 provides a flow chart summarizing daily oral dosing of odd chainsaturated fatty acids and very long even chain saturated fatty acidsprovided to dolphins and resultant serum fatty acid concentrationsachieved that were predictive of raised red blood cell indices andalleviated anemia in dolphins on a modified fish diet.

DETAILED DESCRIPTION

Compositions including one or more fatty acids, and associated methodsfor treatment of anemic conditions are provided.

Anemia is the most common blood disorder in the United States, affectingover 3 million people a year. Anemia is independently associated withpoorer health outcomes in people with chronic diseases, including anincreased risk of developing cardiac disease and an increased risk ofmortality in end stage renal disease patients. The annual cost ofunderlying diseases for people with concurrent anemia of chronic disease(ACD) is double that of people with chronic diseases without ACD. Thus,anemic diseases are implicated in a widespread public health problem.

Generally, anemia is indicated by detection of low hemoglobin, lowhematocrit, low packed cell volume, and/or the oxygen-carrying capacityof the blood being low. A number of subconditions have been identified.Types of anemia include inherited forms such as thalassemia and sicklecell anemia, those in which red blood cell (RBC) production is low,those in which red blood cells are abnormal in shape or size, and thosein which red blood cells undergo elevated rates of cell death. Symptomsof anemia include fatigue, weakness, pale or yellowish skin, irregularheartbeats, shortness of breath, dizziness or lightheadedness, chestpain, cold hands and feet, and headache. Anemia can further beassociated with the risk of developing heart problems, severe fatigue,pregnancy complications, and death. Anemia can impair cognitivedevelopment and reduce physical work capacity. Risk for anemicconditions is associated with a diet lacking in certain vitamins,intestinal disorders, menstruation, pregnancy, family history of anemia,blood diseases, autoimmune disorders, alcoholism, exposure to toxicchemicals, chronic inflammation, the use of some medications, and poorhealth arising from chronic conditions.

Anemia of chronic disease, also called anemia of inflammation andinflammatory anemia, is anemia associated with a chronic underlyingcondition. The underlying condition, which may be, for example,infections, inflammation, or cancer, may reduce oxygen-carrying capacityby a number of mechanisms, generally related to reducing the populationof circulating RBCs. Generally, ACD develops and presents slowly, withmild or no symptoms.

No specific treatment exists for ACD. Generally, practitioners may treatthe underlying disorder. Erythropoietin or darbepoietin, drugs thatstimulate the bone marrow to produce red blood cells, may beadministered. In certain cases, blood transfusions may be indicated.

It is an object of certain of the embodiments to provide a method fordetecting protective factors for and risk factors against anemicconditions in mammal subjects, such as dolphins and humans. An object ofcertain of the embodiments is to provide a method for treating anemicconditions in mammal subjects, such as dolphins and humans. An object ofcertain of the embodiments is to provide a method for detecting ananemic condition in mammal subjects, such as for dolphins and humans. Itis an object of certain of the embodiments to provide a method forincreasing the serum, plasma, or erythrocyte membrane level of one ormore fatty acids or fatty acid derivatives, including but not limited toodd chain fatty acids, for example, heptadecanoic acid, and/or certaineven chain fatty acids, such as behenic acid, in mammal subjects, suchas dolphins and humans. An object of certain of the embodiments is toprovide a fatty acid supplement or pharmaceutical composition fortreating or preventing an anemic condition. An object of certain of theembodiments is to provide a method for detecting and/or treating anemicconditions in mammal subjects, such as dolphins and humans, that is easyto accomplish in a cost-effective manner.

An object of certain of the embodiments is to provide a method forprophylaxis of an anemic condition in mammal subjects, such as dolphinsand humans.

An object of certain of the embodiments is to provide a method forincreasing an odd chain fatty acid in the sera, plasma, or erythrocytemembranes of mammal subjects, such as dolphins and humans. An object ofcertain of the embodiments is to provide a method for increasing verylong even chain fatty acids in the sera of mammal subjects, such asdolphins and humans. An object of certain of the embodiments is toprovide an odd chain fatty acid substantially free from other fattyacids in mammal subjects, such as dolphins and humans. An object ofcertain of the embodiments is to provide one or more odd chain fattyacids substantially free from even chain fatty acids in mammal subjects,such as dolphins and humans.

It is an object of certain of the embodiments is to provide a method fordetecting and treating an anemic condition in mammal subjects, such asdolphins and humans. An object of certain of the embodiments is toprovide a fatty acid, such as an odd chain fatty acid or a very longeven chain fatty acid, for treating an anemic condition in mammalsubjects, such as dolphins and humans. An object of certain of theembodiments is to provide a method for prophylaxis of an anemiccondition in mammal subjects, such as dolphins and humans. An object ofcertain of the embodiments is to provide a method for detecting ortreating a condition associated with oxidative stress in mammalsubjects, such as dolphins and humans. An object of certain of theembodiments is to provide a behenic acid and/or heptadecanoic acidsupplement for treating anemia in mammal subjects, such as dolphins andhumans.

An object of certain of the embodiments is to provide a bioavailableform of odd chain and very long even chain fatty acids to mammalsubjects, such as dolphins and humans. An object of certain of theembodiments is to provide one or more odd chain fatty acids with one ormore certain even chain fatty acids to mammal subjects, such as dolphinsand humans. An object of certain embodiments is to provide a method forincreasing both an odd chain fatty acid and certain even chain fattyacids in the sera of mammal subjects, such as dolphins and humans. Anobject of certain of the embodiments is to provide a method for fattyacid elongation in the sera, plasma, or erythrocyte membranes of mammalsubjects, such as dolphins and humans. An object of certain of theembodiments is to provide a method for fatty acid chain shortening inthe sera, plasma, or erythrocyte membranes of mammal subjects, such asdolphins and humans. An object of certain of the embodiments is toprovide a method for altering concentrations of a variety of odd chainand very long even chain fatty acid forms, including neutral forms (e.g.free fatty acids, cholesterol esters, diacylglycerides, andtriacylglycerides), phospholipids (e.g. phosphatidylcholine,phosphatidylethanolamine, lysophosphatidylcholine, andlysophosphatidylethanolamine), and sphingolipids (e.g. ceramides,hexosylceramides, and sphingosines) in the sera, plasma, or erythrocytemembranes of mammal subjects, such as dolphins and humans.

Compositions including one or more of certain even chain fatty acids,and associated methods for treatment of an anemic condition areprovided. Compositions including one or more bioavailable even chainfatty acids are provided.

One or more than one of the aforementioned objects is provided by orachieved by the various compositions, methods, and uses as describedherein.

Definitions

The term “alcohol” as used herein is a broad term, and is to be givenits ordinary and customary meaning to a person of ordinary skill in theart (and is not to be limited to a special or customized meaning), andrefers without limitation to any compound as described hereinincorporating one or more hydroxy groups, or being substituted by orfunctionalized to include one or more hydroxy groups.

The term “derivative” as used herein is a broad term, and is to be givenits ordinary and customary meaning to a person of ordinary skill in theart (and is not to be limited to a special or customized meaning), andrefers without limitation to any compound as described hereinincorporating one or more derivative groups, or being substituted by orfunctionalized to include one or more derivative groups. Derivativesinclude but are not limited to esters, amides, anhydrides, acid halides,thioesters, phosphates, triphosphates, and 3-sulfenyl derivatives.

The term “hydrocarbon” as used herein is a broad term, and is to begiven its ordinary and customary meaning to a person of ordinary skillin the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to any moiety comprising onlycarbon and hydrogen atoms. A functionalized or substituted hydrocarbonmoiety has one or more substituents as described elsewhere herein.

The term “lipid” as used herein is a broad term, and is to be given itsordinary and customary meaning to a person of ordinary skill in the art(and is not to be limited to a special or customized meaning), andrefers without limitation to saturated and unsaturated oils and waxes,derivatives, amides, glycerides, fatty acids, fatty alcohols, sterol andsterol derivatives, phospholipids, ceramides, sphingolipids,tocopherols, and carotenoids, among others.

The terms “pharmaceutically acceptable” as used herein is a broad term,and is to be given its ordinary and customary meaning to a person ofordinary skill in the art (and is not to be limited to a special orcustomized meaning), and refers without limitation to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for contact with the tissues ofand/or for consumption by human beings and animals without excessivetoxicity, irritation, allergic response, or other problem complicationscommensurate with a reasonable risk/benefit ratio.

The terms “pharmaceutically acceptable salts” and “a pharmaceuticallyacceptable salt thereof” as used herein are broad terms, and are to begiven their ordinary and customary meaning to a person of ordinary skillin the art (and is not to be limited to a special or customizedmeaning), and refer without limitation to salts prepared frompharmaceutically acceptable, non-toxic acids or bases. Suitablepharmaceutically acceptable salts include metallic salts, e.g., salts ofaluminum, zinc, alkali metal salts such as lithium, sodium, andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts; organic salts, e.g., salts of lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine), procaine, and tris;salts of free acids and bases; inorganic salts, e.g., sulfate,hydrochloride, and hydrobromide; and other salts which are currently inwidespread pharmaceutical use and are listed in sources well known tothose of skill in the art, such as, for example, The Merck Index. Anysuitable constituent can be selected to make a salt of the therapeuticagents discussed herein, provided that it is non-toxic and does notsubstantially interfere with the desired activity. In addition to salts,pharmaceutically acceptable precursors and derivatives of the compoundscan be employed. Pharmaceutically acceptable amides, lower alkylderivatives, and protected derivatives can also be suitable for use incompositions and methods of preferred embodiments. While it may bepossible to administer the compounds of the preferred embodiments in theform of pharmaceutically acceptable salts, it is generally preferred toadminister the compounds in neutral form.

The term “pharmaceutical composition” as used herein is a broad term,and is to be given its ordinary and customary meaning to a person ofordinary skill in the art (and is not to be limited to a special orcustomized meaning), and refers without limitation to a mixture of oneor more compounds disclosed herein with other chemical components, suchas diluents or carriers. The pharmaceutical composition facilitatesadministration of the compound to an organism. Pharmaceuticalcompositions can also be obtained by reacting compounds with inorganicor organic acids or bases. Pharmaceutical compositions will generally betailored to the specific intended route of administration.

As used herein, a “carrier” as used herein is a broad term, and is to begiven its ordinary and customary meaning to a person of ordinary skillin the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to a compound that facilitatesthe incorporation of a compound into cells or tissues. For example,without limitation, dimethyl sulfoxide (DMSO) is a commonly utilizedcarrier that facilitates the uptake of many organic compounds into cellsor tissues of a subject. Water, saline solution, ethanol, and mineraloil are also carriers employed in certain pharmaceutical compositions.

As used herein, a “diluent” as used herein is a broad term, and is to begiven its ordinary and customary meaning to a person of ordinary skillin the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to an ingredient in apharmaceutical composition that lacks pharmacological activity but maybe pharmaceutically necessary or desirable. For example, a diluent maybe used to increase the bulk of a potent drug whose mass is too smallfor manufacture and/or administration. It may also be a liquid for thedissolution of a drug to be administered by injection, ingestion orinhalation. A common form of diluent in the art is a buffered aqueoussolution such as, without limitation, phosphate buffered saline thatmimics the composition of human blood.

As used herein, an “excipient” as used herein is a broad term, and is tobe given its ordinary and customary meaning to a person of ordinaryskill in the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to a substance that is added toa pharmaceutical composition to provide, without limitation, bulk,consistency, stability, binding ability, lubrication, disintegratingability etc., to the composition. A “diluent” is a type of excipient.

As used herein, a “subject” as used herein is a broad term, and is to begiven its ordinary and customary meaning to a person of ordinary skillin the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to an animal that is the objectof treatment, observation or experiment. “Animal” includes cold- andwarm-blooded vertebrates and invertebrates such as fish, shellfish,reptiles and, in particular, mammals. “Mammal” includes, withoutlimitation, dolphins, mice, rats, rabbits, guinea pigs, dogs, cats,sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, andapes, and, in particular, humans. In some embodiments, the subject ishuman.

As used herein, the terms “treating,” “treatment,” “therapeutic,” or“therapy” are broad terms, and are to be given their ordinary andcustomary meaning (and are not to be limited to a special or customizedmeaning) and, without limitation, do not necessarily mean total cure orabolition of the disease or condition. Any alleviation of any undesiredmarkers, signs or symptoms of a disease or condition, to any extent, canbe considered treatment and/or therapy. Furthermore, treatment mayinclude acts that may worsen the patient's overall feeling of well-beingor appearance.

The terms “therapeutically effective amount” and “effective amount” asused herein are broad terms, and are to be given its ordinary andcustomary meaning to a person of ordinary skill in the art (and are notto be limited to a special or customized meaning), and are used withoutlimitation to indicate an amount of an active compound, orpharmaceutical agent, that elicits the biological or medicinal responseindicated. For example, a therapeutically effective amount of compoundcan be the amount needed to prevent, alleviate or ameliorate markers orsymptoms of a condition or prolong the survival of the subject beingtreated. This response may occur in a tissue, system, animal or humanand includes alleviation of the signs or symptoms of the disease beingtreated. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, in view of thedisclosure provided herein. The therapeutically effective amount of thecompounds disclosed herein required as a dose will depend on the routeof administration, the type of animal, including human, being treated,and the physical characteristics of the specific animal underconsideration. The dose can be tailored to achieve a desired effect, butwill depend on such factors as weight, diet, concurrent medication andother factors which those skilled in the medical arts will recognize.

The term “solvents” as used herein is a broad term, and is to be givenits ordinary and customary meaning to a person of ordinary skill in theart (and is not to be limited to a special or customized meaning), andrefers without limitation to compounds with some characteristics ofsolvency for other compounds or means, that can be polar or nonpolar,linear or branched, cyclic or aliphatic, aromatic, naphthenic and thatincludes but is not limited to: alcohols, derivatives, diesters,ketones, acetates, terpenes, sulfoxides, glycols, paraffins,hydrocarbons, anhydrides, heterocyclics, among others.

Any percentages, ratios or other quantities referred to herein are on aweight basis, unless otherwise indicated.

Odd Chain Fatty Acids and Very Long Even Chain Fatty Acids

Fatty acids include saturated and unsaturated fatty acids As providedherein, fatty acids are referred to and described using conventionalnomenclature as is employed by one of skill in the art. A saturatedfatty acid includes no carbon-carbon double bonds. An unsaturated fattyacid includes at least one carbon-carbon double bond. A monounsaturatedfatty acid includes only one carbon-carbon double bond. Apolyunsaturated fatty acid includes two or more carbon-carbon doublebonds. Double bonds in fatty acids are generally cis; however, transdouble bonds are also possible. The position of double bonds can beindicated by Δn, where n indicates the lower numbered carbon of eachpair of double-bonded carbon atoms. A shorthand notation in a form total# carbons:# double bonds, Δ_(double bond positions) can be employed. Forexample, 20:4Δ_(5,8,11,14) refers to a fatty acid having 20 carbon atomsand four double bonds, with the double bonds situated between the 5 and6 carbon atom, the 8 and 9 carbon atom, the 11 and 12 carbon atom, andthe 14 and 15 carbon atom, with carbon atom 1 being the carbon of thecarboxylic acid group. Stearate (octadecanoate) is a saturated fattyacid. Oleate (cis-Δ9-octadecenoate) is a monounsaturated fatty acid,linolenate (all-cis-Δ9,12,15-octadecatrienoate) is a polyunsaturatedfatty acid. The total number of carbons can be preceded by “C” anddouble bond positions can be unspecified, e.g., C20:4 referring to afatty acid having 20 carbon atoms and four double bonds.

A fatty acid may be referred to by various names, for example,heptadecanoic acid may be referred to as heptadecylic acid, margaricacid, and n-heptadecylic acid, or C17:0. A fatty acid may be referred toby lipid numbers, as known in the art.

In some embodiments, the fatty acid can be an odd chain fatty acid or avery long even chain fatty acid. In further embodiments, one or morefatty acids can include at least one odd chain fatty acid or at leastone very long even chain fatty acid.

Examples of odd chain fatty acids are margaric acid (heptadecanoic acid,C17:0), pelargonate (nonanoic acid, C9:0), undecanoic acid (C11:0),nonadecanoic acid (C19:0), pentadecanoic acid (C15:0), arachidonate((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoic acid), adrenate(all-cis-7,10,13,16-docosatetraenoic acid), and osbond acid(all-cis-4,7,10,13,16-docosapentaenoic acid). Generally, the one or moreodd chain fatty acids have from 9 carbon atoms to 31 carbon atoms (9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31 carbon atoms), forexample, from 15 to 21 carbon atoms, for example 17 carbon atoms;however, in certain embodiments higher or lower odd numbers of carbonatoms can be acceptable. Generally, the one or more odd chain fattyacids are saturated; however, in certain embodiments mono orpolyunsaturated odd chain fatty acids can be acceptable.

An odd chain fatty acid may include saturated or unsaturated hydrocarbonchains. An odd chain fatty acid may be present as a carboxylicderivative. An odd chain fatty acid may be present as a salt, forexample, at the carboxylic group. In some embodiments, one odd chainfatty acid may be present, two odd chain fatty acids may be present,three odd chain fatty acids may be present, or more. In someembodiments, odd chain fatty acids in a mixture including a plurality ofodd chain fatty acids may be distinguished by the amount ofunsaturation, the length of the hydrocarbon chain, varying states ofderivativeification, or by other structural features.

Odd chain fatty acids are found in trace amounts in some dairy products,including butter, and is a component of some fish (see, e.g., Mansson HL (2008), Fatty acids in bovine milk fat, Food Nutr. Res. 52:4, Luzia LA, Sampaio G R, Castellucci C M N, Torres E A F S (2013) The influenceof season on the lipid profiles of five commercially important speciesof Brazilian fish. Food Chem. 83:93-97). Studies have demonstrated thatincreasing daily dietary intake of foods with odd chain fatty acidssuccessfully increases serum or plasma levels (see, e.g., Benatar J. R.,Stewart R. A. H. (2014), The effects of changing dairy intake on transand saturated fatty acid levels—results from a randomized controlledstudy. Nutr. J. 13:32).

As used herein, the term “very long” as applied to fatty acids, such aseven chain fatty acids, refers to those fatty acids having at least 20carbon atoms, e.g., fatty acids having from 20 to 26 carbon atoms, forexample, behenic acid (C22:0).

The one or more very long even chain fatty acids can have, for example,20, 22, 24, 26, 28, or 30 carbon atoms; however, in certain embodimentshigher or lower even numbers of carbon atoms can be acceptable.Generally, the one or more very long even chain fatty acids aresaturated; however, in certain embodiments mono or polyunsaturated verylong even chain fatty acids can be acceptable. A very long even chainfatty acid may be present as a carboxylic derivative. A very long evenchain fatty acid may be present as a salt, for example, at thecarboxylic group. In some embodiments, one very long even chain fattyacid may be present, two very long even chain fatty acids may bepresent, three very long even chain fatty acids may be present, or more.

In some embodiments, very long even chain fatty acids in a mixtureincluding a plurality of very long even chain fatty acids may bedistinguished by the amount of unsaturation, the length of thehydrocarbon chain, varying states of derivativeification, or by otherstructural features. The very long even chain fatty acid may be providedin a bioavailable form.

Very long even chain fatty acids are found in trace amounts in pracaxioil, derived from the seeds of the Pentaclethra macroloba tree, and benoil, derived from the seeds of Moringa oleifera. A pure or purified verylong even chain fatty acid may exist in various physical states. Forexample, behenic acid exists as a white to cream color powder orcrystals that is stable at room temperature. Behenic acid can bepurchased in forms suitable for research purposes in small amounts fromsome commercial suppliers (for example, from Sigma-Aldrich corp., of St.Louis, Mo.). Other very long even chain fatty acids, or salts orderivatives thereof, may exist as oils, solids, crystalline solids.

Generally, a fatty acid, such as an odd chain fatty acid or a very longeven chain fatty acid can be provided as a free fatty acid, or aderivative thereof. Such derivatives include, but are not limited to,acyl glycerides. An acyl glyceride may be substituted with up to threeacyl fatty acid esters. Thus, an acyl glyceride can be amonoacylglyceride (MAG), diacylglyceride (DAG), or a triacylglyceride(TAG). The glyceride can include more than one type of fatty acid ester.For example, a glyceride can include a heptdecanoate and a docosanoate.A glyceride can also be a structured triacylglyceride (STAG), aplasmalogen, or a phospholipid. The fatty acid ester can be in the sn1position or the sn2 position, or both positions. The sn1 and sn2positions can be substituted by the same or different fatty acid esters.As a non-limiting example, a structured triacylglyceride can besn-1,3-C17-sn-2-oleoyl.

In some embodiments, a fatty acid can be provided as a free fatty acid,a cholesterol ester, a glycerol ester (including, but not limited to amonoacylglyceride (MAG), diacylglyceride (DAG), or a triacylglyceride(TAG)), a phospholipid (including, but not limited to, aphosphatidylcholine, a lysophosphatidylcholine, aphosphatidylethanolamine, a lysophosphatidylethanolamine, or aphosphatidylserine), a ceramide (including but not limited to a hexosylceramide) or a sphingolipid. A non-limiting example of aphophatidylcholine is 2,3-di-C17:0-phosphatidylcholine. A non-limitingexample of a lysophophatidylcholine is2-lyso-3-C17:0-phosphatidylcholine. In some embodiments, a derivative ofa fatty acid can be a β-sulfenyl derivative. It is thought thatβ-sulfenyl derivative, such as an acid or ester, can be resistant toβ-oxidation in the body. As a non-limiting example, the β-sulfenylderivative of heptadecanoic acid is tetradecylthioacetic acid.Derivatives can be synthesized by standard methods known to those ofskill in the art.

In some embodiments, a fatty acid may be provided as a constituent of aspecific type of lipid, for example, a ceramide, a phospholipid, asphingolipid, a membrane lipid, a glycolipid, or a triglyceride.

In some embodiments, a fatty acid, such as a very long even chain fattyacid, is provided in a bioavailable form. The term “bioavailability”refers to the fraction of an administered dose of unchanged drug thatreaches the systemic circulation, one of the principal pharmacokineticproperties of drugs. By definition, when a medication is administeredintravenously, its bioavailability is 100%. As employed herein, the term“bioavailable” refers to a form of the fatty acid that is successfullyabsorbed by the body when using methods of administration other thanintravenous, for example, an oral therapeutic). In some embodiments,very long even chain fatty acid-based compositions may include adaptionsthat optimize absorption. In some embodiments, a very long even chainfatty acid can be provided as a structured triacylglyceride. In furtherembodiments, the fatty acid is in the sn-2 position of a structuredtriacylglyceride.

A pure or purified fatty acid may exist in various physical states. Forexample, heptadecanoic acid exists as an off-white powder that is stableat room temperature; this compound can be purchased in forms suitablefor research purposes in small amounts from some commercial suppliers(for example, from Sigma-Aldrich corp., of St. Louis, Mo.). Other fattyacids, or salts or derivatives thereof, may exist as oils, solids,crystalline solids, or gases.

An odd chain fatty acid or a very long even chain fatty acid, or thepharmaceutically acceptable salts or derivatives thereof, may beprovided in a purity (e.g., a percentage of the fatty acid, or itspharmaceutically acceptable salts or derivatives, in a bulk form) of atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about98%, at least about 99%, at least about 99.9%, at least about 99.99%, orsubstantially pure, wherein substantially pure may include, but not belimited to, a product with impurities at a level such that nophysiological effect from the presence of the impurities is detectable.A mixture of fatty acids, such as, for example, odd chain fatty acidsand/or very long even chain fatty acids, or pharmaceutically acceptablesalts or derivatives thereof, may be present in a purity of at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, at least about 99.9%, at least about 99.99%, orsubstantially pure. The fatty acid, or a mixture thereof, or apharmaceutically acceptable salt or derivative thereof, may be free fromother fatty acids or fatty acid derivatives, may be free fromtriglycerides, or may be free from phospholipids. Without limitation, anodd chain fatty acid as provided herein may be substantially free fromeven chain fatty acids, singly or taken as a group; even chain fattyacids include, for example, myristic acid (C14:0), palmitic acid(C16:0), or stearic acid (C18:0). In some embodiments, an odd chainfatty acid as provided herein may be substantially free from short-chainfatty acids (SCFA), medium-chain fatty acids (MCFA), long-chain fattyacids (LCFA), or very long chain fatty acids (VLCFA).

A fatty acid, such as an odd chain fatty acid or a very long even chainfatty acid, or a pharmaceutically acceptable salt or derivative thereof,may be from any source. In some embodiments, a fatty acid, or itspharmaceutically acceptable salts or derivatives, may be present innatural sources, may be isolated from natural sources, may besemi-synthetic, may be synthetic, or may be a mixture of one or more ofthese. The fatty acid, or its pharmaceutically acceptable salts orderivatives, may be produced in a laboratory, may be produced in nature,may be produced by enzymatic processes, may be produced by wildmicrobes, may be produced by genetically modified microbes, may beisolated from animal tissues, may be produced by chemical synthesis, ormay be produced by a plurality of these processes.

The fatty acid may be derived from natural sources, e.g., fish oils, orcan be synthesized by methods as are known in the art. In someembodiments, the fatty acid may be contaminated with undesiredcomponents present in unrefined or unpurified natural products. In suchsituations, it can be desirable to remove undesired components, or toincrease the concentration of desired components using known separationor purification techniques.

In any compound described, all tautomeric forms are also intended to beincluded. Without limitation, all tautomers of carboxylic groups areintended to be included.

In any compound described herein having one or more double bond(s)generating geometrical isomers that can be defined as E or Z, eachdouble bond may independently be E or Z, or a mixture thereof.

Where compounds disclosed herein have unfilled valencies, then thevalencies are to be filled with hydrogens or isotopes thereof, e.g.,hydrogen-1 (protium) and hydrogen-2 (deuterium).

The fatty acid, such as an odd chain fatty acid or a very long evenchain fatty acid, as described herein, includes crystalline forms (alsoknown as polymorphs, which include the different crystal packingarrangements of the same elemental composition of a compound), amorphousphases, salts, solvates, and hydrates. In some embodiments, thecompounds described herein exist in solvated forms with pharmaceuticallyacceptable solvents such as water, ethanol, or the like. In otherembodiments, the compounds described herein exist in unsolvated form.Solvates contain either stoichiometric or non-stoichiometric amounts ofa solvent, and may be formed during the process of crystallization withpharmaceutically acceptable solvents such as water, ethanol, or thelike. Hydrates are formed when the solvent is water, or alcoholates areformed when the solvent is alcohol. In addition, the compounds providedherein can exist in unsolvated as well as solvated forms. In general,the solvated forms are considered equivalent to the unsolvated forms forthe purposes of the compounds and methods provided herein.

The compounds described herein can be labeled isotopically. In somecircumstances, substitution with isotopes such as deuterium may affordcertain therapeutic advantages resulting from greater metabolicstability, such as, for example, increased in vivo half-life or reduceddosage requirements. Isotopic substitution may be beneficial inmonitoring subject response to administration of a compound, forexample, by providing opportunity for monitoring of the fate of an atomin a compound. Each chemical element as represented in a compoundstructure may include any isotope of said element. For example, in acompound structure a hydrogen atom may be explicitly disclosed orunderstood to be present in the compound. At any position of thecompound that a hydrogen atom may be present, the hydrogen atom can beany isotope of hydrogen, including but not limited to hydrogen-1(protium) and hydrogen-2 (deuterium). Thus, reference herein to acompound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

The prevalence of various fatty acids in the diet has been correlated tothe occurrence of metabolic syndrome in subjects (see, e.g., Forouhi N,Koulman A, Sharp S, Imamura F, Kroger J, Schulze M, et al. (2014),Differences in the prospective association between individual plasmaphospholipid saturated fatty acids and incident type 2 diabetes: theEPIC-InterAct case-cohort study. Lancet Diabetes Endocrinol. 2:810-8).Indeed, whole-fat dairy consumption has been correlated with a decreasedrisk of metabolic syndrome markers (see, e.g., Kratz M, Marcovina S,Nelson J E, Yeh M M, Kowdley K V, Callahan H S, et al. (2014), Dairy fatintake is associated with glucose tolerance, hepatic and systemicinsulin sensitivity, and liver fat but not beta-cell function in humans,Am. J. Clin. Nutr., 99:1385-96).

The mechanism(s) by which fatty acid(s) have a beneficial effect are notwell understood. Without wishing to be limited by theory, it is thoughtthat fatty acids, or derivatives thereof, can be elongated (increased inchain length) or chain shortened by metabolic processes in the body, toform different fatty acids, or derivatives thereof. Peroxidation ofcertain fatty acids may create products with signaling characteristicsin the body. It is thought that fatty acids of certain chain lengthcreate signaling products that substantially contribute to one or moreconditions provided herein. In some embodiments, an odd chain fatty acidis elongated to form a very long chain fatty acid, such as a very longeven chain fatty acid. In further embodiments, a very long even chainfatty acid can be chain-shortened to an odd chain fatty acid. Levels ofvery long even chain fatty acids in the body may increase followingadministration of one or more odd chain fatty acids. Levels of odd chainfatty acids in the body may increase following administration of one ormore very long even chain fatty acids.

Pharmaceutical Compositions Including One or More Fatty Acids

Formulations including a fatty acid, such as an odd chain fatty acid ora very long even chain fatty acid, or a salt or derivative thereof, andat least one excipient are provided. It is generally preferred toadminister the compounds of the embodiments in oral formulations;however, other routes of administration are also contemplated.

The pharmaceutical compositions described herein can be administered bythemselves to a subject, or in compositions where they are mixed withother active agents, as in combination therapy, or with carriers,diluents, excipients or combinations thereof. Formulation is dependentupon the route of administration chosen. Techniques for formulation andadministration of the compounds described herein are known to thoseskilled in the art (see, e.g., “Remington: The Science and Practice ofPharmacy”, Lippincott Williams & Wilkins; 20th edition (Jun. 1, 2003)and “Remington's Pharmaceutical Sciences,” Mack Pub. Co.; 18th and 19theditions (December 1985, and June 1990, respectively).

The pharmaceutical compositions disclosed herein may be manufactured bya process that is itself known, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, tableting, or extracting processes. Many ofthe compounds used in the pharmaceutical combinations disclosed hereinmay be provided as salts with pharmaceutically acceptable counterions.

Multiple techniques of administering a compound exist in the artincluding, but not limited to, oral, rectal, topical, aerosol, injectionand parenteral delivery, including intramuscular, subcutaneous,intravenous, intramedullary injections, intrathecal, directintraventricular, intraperitoneal, intranasal and intraocularinjections. Contemplated herein is any combination of the forgoing, orother methods as would be known to one of ordinary skill in the art(see, e.g., “Remington: The Science and Practice of Pharmacy”,Lippincott Williams & Wilkins; 20th edition (Jun. 1, 2003) and“Remington's Pharmaceutical Sciences,” Mack Pub. Co.; 18th and 19theditions (December 1985, and June 1990, respectively).

In practice, a fatty acid, such as an odd chain fatty acid or a verylong even chain fatty acid, or a salt or derivative thereof, may becombined as the active ingredient in intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier can take a wide variety of formsdepending on the form of preparation desired for administration. Thus,the pharmaceutical compositions provided herein can be presented asdiscrete units suitable for oral administration such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient. Further, the compositions can be presented as an oil, apowder, as granules, as a solution, as a suspension in an aqueousliquid, as a non-aqueous liquid, as an oil-in-water emulsion, or as awater-in-oil liquid emulsion. In addition to the common dosage forms setout above, the compounds provided herein, or pharmaceutically acceptablesalts or derivatives thereof, can also be administered by controlledrelease means and/or delivery devices. The compositions can be preparedby any of the methods of pharmacy. In general, such methods include astep of bringing into association the active ingredient with the carrierthat constitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both. The product can then be conveniently shaped into the desiredpresentation.

A formulation may also be administered in a local rather than systemicmanner, for example, via injection of the compound directly into theinfected area, often in a depot or sustained release formulation.Furthermore, a targeted drug delivery system might be used, for example,in a liposome coated with a tissue specific antibody.

The pharmaceutical compositions may contain a fatty acid, such as an oddchain fatty acid or a very long even chain fatty acid, or a salt orderivative thereof, in an amount effective for the desired therapeuticeffect. In some embodiments, the pharmaceutical compositions are in aunit dosage form and comprise from about 0.1 mg or less to about 5000 mgor more per unit dosage form. In further embodiments, the pharmaceuticalcompositions comprise from about 1 to about 500 mg per unit dosage formor from about 500 to 5000 mg per unit dosage form. Such dosage forms maybe solid, semisolid, liquid, an emulsion, or adapted for delivery viaaerosol or the like for inhalation administration.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, lower alcohols, and water. Examples of gaseous carriersinclude carbon dioxide and nitrogen.

Pharmaceutical compositions provided herein can be prepared as solutionsor suspensions of the active compound(s) in water. A suitable surfactantcan be included such as, for example, hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof in oils. Further, a preservative can beincluded to, for example, prevent the detrimental growth ofmicroorganisms.

Pharmaceutical compositions provided herein suitable for injectable useinclude sterile aqueous solutions or dispersions. Furthermore, thecompositions can be in the form of sterile powders for theextemporaneous preparation of such sterile injectable solutions ordispersions. The pharmaceutical compositions must be stable under theconditions of manufacture and storage; thus, preferably should bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol and liquid polyethylene glycol), vegetable oils, andsuitable mixtures thereof.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above can include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining a compound provided herein, or pharmaceutically acceptablesalt or derivative thereof, can also be prepared in powder or liquidconcentrate form for dilution.

The fatty acid, such as an odd chain fatty acid or a very long evenchain fatty acid, or a salt or derivative thereof, can be formulated asa liposome. The fatty acid can be a component of the lipid portion ofthe liposome or can be encapsulated in the aqueous portion of theliposome. The fatty acid, such as an odd chain fatty acid or a very longeven chain fatty acid, or a salt or derivative thereof, can also becoformulated with a cyclodextrin. The cyclodextrin can be, for example,hydroxypropyl-β-cyclodextrin or a sulfobutylether cyclodextrin.

Contemplated herein are compositions including a fatty acid, such as anodd chain fatty acid or a very long even chain fatty acid, or a salt orderivative thereof in combination with at least one additional activeagent. A fatty acid, such as an odd chain fatty acid or a very long evenchain fatty acid, or a salt or derivative thereof, and the at least oneadditional active agent(s) may be present in a single formulation or inmultiple formulations provided together, or may be unformulated (forexample, free of excipients and carriers). In some embodiments, a fattyacid, such as an odd chain fatty acid or a very long even chain fattyacid, or a salt or derivative thereof, can be administered with one ormore additional agents together in a single composition. For example, acompound of a fatty acid, such as an odd chain fatty acid or a very longeven chain fatty acid, or a salt or derivative thereof, can beadministered in one composition, and at least one of the additionalagents can be administered in a second composition. In a furtherembodiment, a fatty acid, such as an odd chain fatty acid or a very longeven chain fatty acid, or a salt or derivative thereof and the at leastone additional active agent(s) are co-packaged in a kit. For example, adrug manufacturer, a drug reseller, a physician, a compounding shop, ora pharmacist can provide a kit comprising a disclosed compound orproduct and another component for delivery to a patient.

Some embodiments described herein relate to a pharmaceuticalcomposition, which can include a therapeutically effective amount of oneor more compounds described herein (e.g., a fatty acid, such as an oddchain fatty acid or a very long even chain fatty acid, or apharmaceutically acceptable salt or derivative thereof) and apharmaceutically acceptable carrier, diluent, excipient or combinationthereof. The pharmaceutical composition can include a fatty acid such asan odd chain fatty acid or a very long even chain fatty acid, or a saltor derivative thereof in, for example, >1%, ≥2%, ≥3%, ≥4%, ≥5%, ≥6%,≥7%, ≥8%, ≥9%, ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%,≥95%, or ≥98% of the composition. In some embodiments, thepharmaceutical composition can include a plurality of fatty acids, suchas one or more of an odd chain fatty acid and/or a very long even chainfatty acid, or salts or derivatives thereof in, for example, >1%, ≥2%,≥3%, ≥4%, ≥5%, ≥6%, ≥7%, ≥8%, ≥9%, ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%,≥70%, ≥80%, ≥90%, ≥95%, or ≥98% of the composition.

Formulations including a very long even chain fatty acid, or a salt orderivative thereof, and an odd chain fatty acid, or a salt or derivativethereof and at least one excipient are provided. It is generallypreferred to administer the compounds of the embodiments in oralformulations; however, other routes of administration are alsocontemplated, as provided herein. In some embodiments, thepharmaceutical composition can include a very long even chain fatty acidand an odd chain fatty acid, or salts or derivatives thereof in, forexample, >1%, ≥2%, ≥3%, ≥4%, ≥5%, ≥6%, ≥7%, ≥8%, ≥9%, ≥10%, ≥20%, ≥30%,≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, or ≥98% of the composition,e.g., from 1% to 98% of the composition, or any amount in between. Aratio of odd chain fatty acid, or a salt or derivative, to very longeven chain fatty acid, or a salt or derivative, present in theformulation, on a weight basis, can be 5:95, 10:90, 15:85, 20:80, 25:75,30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25,80:20, 85:15, 90:10, or 95:5. In some embodiments, the fatty acidspresent in the formulation comprise 25% to 75% odd chain fatty acid(s),or salts or derivatives, with the remainder comprising very long evenchain fatty acid(s), or salts or derivatives.

Foodstuffs

Foodstuffs and other comestibles including a fatty acid, such as an oddchain fatty acid or a very long even chain fatty acid, or a salt orderivative thereof, are provided, wherein an amount of the fatty acid inthe foodstuff has been fortified (e.g., enriched or concentrated). Afatty acid, such as an odd chain fatty acid or a very long even chainfatty acid, provided herein may be added to foodstuffs for consumptionby a subject. The fatty acid, such as an odd chain fatty acid or a verylong even chain fatty acid, may be integrated into one or moreingredients of a foodstuff. The fatty acid, such as an odd chain fattyacid or a very long even chain fatty acid, may be prepared as aningredient, or may be unprepared. The compound, or preparation includingthe compound, may be added prior to preparation, during preparation, orfollowing preparation. Preparation may without limitation includecooking, mixing, flavoring, seasoning, blending, boiling, frying,baking, or other processes known in the art. Fortification is preferablyat a level so as to provide a therapeutic daily dosage of the fatty acidas described elsewhere herein; however, beneficial effects may also beobtained at amounts below such dosages.

A fatty acid, such as an odd chain fatty acid or a very long even chainfatty acid, or salt or derivative thereof, as provided herein may bepresent as a constituency in foodstuffs by operation of processes knownin nature, for example, by altering the metabolic processes of a plant,animal, bacteria, or fungus. Genetic alteration of a plant, animal,bacteria, or fungus to increase the concentration of a fatty acid, suchas an odd chain fatty acid or a very long even chain fatty acid, or asalt or derivative thereof, is contemplated. By way of example, thefatty acid can be present in the foodstuff in a concentration of atleast about 1%, at least about 2%, at least about 3%, at least about 4%,at least about 5%, at least about 6%, at least about 7%, at least about8%, at least about 9%, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, or higher, forexample, 1% to 2% or 3% or 4% or 5% or 6% or 7% or 8% or 9% or 10% or20% or 30% or 40% or 50%.

Indications

Provided are compositions and methods for treating conditions related toanemia and anemic conditions. These conditions include but are notlimited to those related to anemic disorders including, but not limitedto hemolytic anemias (including but not limited to thalassemias,hereditary spherocytosis, hereditary elliptocytosis, glucose-6-phosphatedehydrogenase deficiency, pyruvate kinase deficiency, immune hemolyticanemia, alloimmune hemolytic anemia, drug-induced hemolytic anemia,mechanical hemolytic anemias, and paroxysmal nocturnal hemoglobinuria),anemia of chronic disease (wherein the underlying condition can be, forexample, autoimmune disorders (such as, for example Crohn disease,systemic lupus erythematosus, rheumatoid arthritis and ulcerativecolitis), neoplastic disorders including cancer (such as, for examplelymphoma and Hodgkin disease), long-term infections (such as, forexample bacterial, viral, and fungal infections), rheumatoid arthritis,ulcerative colitis, Hodgkin disease, metabolic syndrome, diabetes (forexample, type 2 diabetes), and other causes of chronic inflammation),anemia, aplastic anemias (including but not limited to congenitalhypoplastic anemia, Diamond-Blackfan anemia and Fanconi anemia), irondeficiency anemia, anemias of abnormal RBC size (including but notlimited to megaloblastic anemia and microcytic anemia), vitamindeficiency anemias (including but not limited to pernicious anemia)anemia of RBC mutation (including but not limited to thalassemia,sideroblastic anemia and sickle cell anemia).

In some embodiments, the compositions and methods provided herein areindicated for treatment, prophylaxis, prevention or maintenance ofanemic conditions.

Anemia as described herein generally relates to a condition in whichthere is low hemoglobin, low hematocrit, low packed cell volume, orreduced oxygen-carrying capacity of red blood cells to tissues. Anemiaalso generally relates to a number of conditions as described herein.Types of anemia include inherited forms such as thalassemia and sicklecell anemia, those in which RBC production is low, those in which thebody produces RBCs of abnormal shape or size, and those in which RBCsundergo elevated rates of cell death.

Anemic conditions include, but are not limited to, hemolytic anemias(including but not limited to thalassemias, hereditary spherocytosis,hereditary elliptocytosis, glucose-6-phosphate dehydrogenase deficiency,pyruvate kinase deficiency, immune hemolytic anemia, alloimmunehemolytic anemia, drug-induced hemolytic anemia, mechanical hemolyticanemias, and paroxysmal nocturnal hemoglobinuria), anemia of chronicdisease, anemia, aplastic anemias (including but not limited tocongenital hypoplastic anemia, Diamond-Blackfan anemia and Fanconianemia), iron deficiency anemia, anemias of abnormal RBC size (includingbut not limited to megaloblastic anemia and microcytic anemia), vitamindeficiency anemias (including but not limited to pernicious anemia) andanemia of RBC mutation (including but not limited to thalassemia,sideroblastic anemia and sickle cell anemia).

An anemic condition can be classified as normochromic, which isgenerally understood to be an anemic condition in which theconcentration of hemoglobin in the RBCs is not pathological, but thereare insufficient numbers of RBCs. An anemic condition can be classifiedas normocytic, which is generally understood to be an anemic conditionin which RBCs are not abnormal in size. Normocytic, normochromic anemiasinclude anemia of chronic disease (ACD) and hemolytic anemia.

Anemia associated with premature RBC destruction is generally referredto as hemolytic anemia. Some forms of hemolytic anemia are inherited andothers are acquired. Hemolytic anemia is associated with conditionsincluding but not limited to autoimmune disorders, infections (such as,for example, hepatitis), cell proliferation disorders (such as, forexample, leukemia and lymphoma), certain medications, sickle cellanemia, and Wiskott-Aldrich syndrome. Additional types of hemolyticanemias include: thalassemias, hereditary spherocytosis, hereditaryelliptocytosis, glucose-6-phosphate dehydrogenase deficiency, pyruvatekinase deficiency, immune hemolytic anemia, alloimmune hemolytic anemia,drug-induced hemolytic anemia, mechanical hemolytic anemias, andparoxysmal nocturnal hemoglobinuria.

RBCs can be removed from circulation due to senescence. Without wishingto be bound by theory, RBCs undergo age-dependent alterations, which mayinclude a decline in metabolic activity, a progressive cell shapetransformation, membrane remodelling, oxidative injury,microvesiculation and exposure of surface removal markers. Thesemodifications may trigger phagocytosis by macrophages, in which RBCs aredestroyed. It is thought that the protease calpain may become activatedin the process, which may be triggered by an increase of cytosoliccalcium. Elevated rates of RBC destruction may lead to an anemiccondition.

Anemia of chronic disease (ACD), also called anemia of inflammation andinflammatory anemia, is anemia associated with a chronic underlyingcondition. An underlying condition may suppress production of red bloodcells in the bone marrow, may decrease the lifespan of red blood cells,or may create problems with how the body uses iron. Generally, ACDdevelops and presents slowly, with mild or no symptoms.

Inflammation may play a role in the pathogenesis of ACD. Without wishingto be bound by theory, it is thought that inflammatory cytokines presentin many chronic diseases, which both lower red blood cell production andraise premature breakdown of senescent cells, leading to anemia. Thus,cytokines and cells of the reticuloendothelial system affect ironhomeostasis, production of erythroid progenitor cells, the production oferythropoietin, and the life span of RBCs, each of which can contributeto an anemic condition. Further, ACD may be associated with abnormalhomeostasis of iron in the body. Increased uptake and retention of ironwithin cells of the reticuloendothelial system may be observed. Insubjects having ACD, ferritin levels may be elevated.

Underlying conditions of ACD include those associated with hemolyticanemia, and conditions including, but not limited to autoimmunedisorders (such as, for example Crohn disease, systemic lupuserythematosus, rheumatoid arthritis and ulcerative colitis), neoplasticdisorders including cancer (such as, for example lymphoma and Hodgkindisease), long-term infections (such as, for example bacterial, viral,and fungal infections), rheumatoid arthritis, ulcerative colitis,Hodgkin disease, metabolic syndrome, diabetes (for example, type 2diabetes), and other causes of chronic inflammation.

Where the underlying condition of ACD is a neoplastic disorder,erythropoiesis can be affected by the infiltration of tumor cells intobone marrow. Tumor cells can produce proinflammatory cytokines and freeradicals that damage erythroid progenitor cells. Bleeding episodes,vitamin deficiencies, hypersplenism, autoimmune hemolysis, renaldysfunction, and radio- and chemotherapeutic interventions can alsoaggravate anemia.

Where the underlying condition is an infection, ACD can be classified asanemia of chronic infection (ACI). Erythropoiesis can be affected by theinfiltration of microorganisms, as seen in various infections, includinghuman immunodeficiency virus infection, hepatitis C, and malaria.

Anemia with chronic kidney disease shares some characteristics of anemiaof chronic disease. Decrease in the production of erythropoietin, due torenal insufficiency and accumulating toxins, may contribute. In patientswith end-stage renal disease, chronic immune activation can arise fromcontact activation of immune cells by dialysis membranes and/or fromfrequent episodes of infection.

Early stage treatment is limited, and may include administration ofsoluble iron. Medications include iron dextran, iron sumalate,polysaccharide iron, ferrus fumarate, carbonyl iron, ferrous aspartoglycinate, heme iron polypeptide, ferrus bisglycinate as can be theadministration of other medicaments such as androgen hormones, folicacid, vitamin B12, vitamin C, succinic acid, niacin, pyridoxine,riboflavin, biotin, thiamine, calcium formateandrogen hormones, such aserythropoietin, folic acid, vitamin B12, vitamin C, succinic acid,niacin, pyridoxine, riboflavin, biotin, thiamine, calcium formate,Aminoxin, Anadrol-50, Chromagen Forte, Epoetin alfa, Epogen, Fe C TabPlus, FeRiva, FeRivaFA, Ferocon, Ferotrin, Ferralet 90, Ferrex 28,Ferrogels Forte, FoliTab 500, Fumatinic, Hematogen Forte, Hemetab,Integra Plus, Irospan 42/6, Lenalidomide, Maxaron Forte, Myferon 150Forte, MyKidz Iron, NovaFerrum, Oxymetholone, Procrit, Proferrin-Forte,Pyridoxine, Repliva 21/7, Revlimid, or Tricon. However, thesemedicaments may lead to undesirable side effects.

Risk factors of anemia include, but are not limited to, a diet lackingin folate and soluble iron menstruation, pregnancy, chronic conditions,genetic conditions, family history and age. Indicators of anemia,individually or in any combination, which may but need not present,include reduced oxygen transport in the blood, fatigue and diminishedphysical capacity, or secondary organ dysfunction or damage, includingheart arrhythmias and heart failure. In some instances, anemia can beidentified by small or pale RBCs. Biomarker indices of anemia includeindices and markers provided herein, and other indications available toclinicians, and include, for example, red blood cell count,reticulocytes, hematocrit or packed cell volume, red blood celldistribution width, mean corpuscular hemoglobin concentration (MCHC),mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH),hemoglobin, white cell count, iron deficiency, serum iron, totaliron-binding capacity, transferrin saturation, zinc protoporphyrin, andserum sTfRs.

Aplastic anemia is associated with low RBC production.

Iron deficiency anemia is associated with a deficiency in iron storessuch that erythropoiesis is inadequate.

Sickle cell anemia may be caused by a mutation which results in mutantsickle hemoglobin. Sideroblastic anemias are a group of disorders withthe common features of mitochondrial iron accumulation in bone marrow,ineffective erythropoiesis, and increased tissue iron levels.

Fanconi anemia refers to a rare genetic disorder associated with a highfrequency of hematological abnormalities and congential anomalies.

Vitamin deficiency anemia refers to a lack of healthy red blood cellscaused by lower than normal amounts of certain vitamins. Vitamins linkedto vitamin deficiency anemia include folate, vitamin B-1, or Vitamin C.

Microcytic anemias are characterized by the production of red bloodcells that are smaller than normal. It is believed that microcyticanemia can be caused by one or more of a lack of globin product(thalassemia), restricted iron delivery to a heme group of hemoglobin,and defects in the synthesis of the heme group.

Provided herein are compositions and methods for treating conditionsincluding but not limited to hemolytic anemias (including but notlimited to thalassemias, hereditary spherocytosis, hereditaryelliptocytosis, glucose-6-phosphate dehydrogenase deficiency, pyruvatekinase deficiency, immune hemolytic anemia, alloimmune hemolytic anemia,drug-induced hemolytic anemia, mechanical hemolytic anemias, andparoxysmal nocturnal hemoglobinuria), anemia of chronic disease, anemia,aplastic anemias (including but not limited to congenital hypoplasticanemia, Diamond-Blackfan anemia and Fanconi anemia), iron deficiencyanemia, anemias of abnormal RBC size (including but not limited tomegaloblastic anemia and microcytic anemia), vitamin deficiency anemias(including but not limited to pernicious anemia) anemia of RBC mutation(including but not limited to thalassemia, sideroblastic anemia andsickle cell anemia).

Serum very long even chain fatty acids levels are correlated withimproved indices for anemia. However, the mechanism by which very longeven chain fatty acids act to inhibit or lessen anemia or markers ofanemia is not presently well understood. The methods and markersprovided herein are not to be construed as limited to any particularmechanism, mode of action, or theory. Accordingly, methods of treating,preventing or ameliorating anemia are provided.

Anemic subjects appropriate for treating using the compositions andmethods provided herein may be identified by the risk factors, indicesand markers provided herein, and by other indications available toclinicians, and are characterized by, e.g., low red blood cell count,reticulocytes, hematocrit/packed cell volume, red blood celldistribution width, and hemoglobin; high white blood cell count, irondeficiency, serum iron, total iron-binding capacity, transferrinsaturation, zinc protoporphyrin, and serum sTfRs. In some instances,anemia can be identified by small or pale RBCs.

In some embodiments, the compositions and methods provided herein areindicated for treatment, prevention and or maintenance of conditions,disorders, diseases and defects associated with hemolytic anemia. Insome embodiments, the compositions and methods provided herein areindicated for treatment, prevention and or maintenance of conditions,disorders, diseases and defects associated with anemia of chronicdisease. In some embodiments, the compositions and methods providedherein are indicated for treatment, prevention and or maintenance ofconditions, disorders, diseases and defects associated with normocyticand/or normochromic anemia.

In some embodiments, the compositions and methods provided herein areindicated for treatment, prophylaxis, prevention or maintenance of ananemic condition.

In some embodiments provided herein, the subject may be a dolphin;however, it is generally contemplated that the methods, uses, andcompositions of the embodiments are applied to humans. Like humansubjects, bottlenose dolphin (Tursiops truncatus) subjects can also besusceptible to anemia, including chronic anemia. The underlying causesof chronic anemia in dolphins are unknown.

In some embodiments, the condition treated is an anemic condition.

In some embodiments, the methods provided herein increase levels ofserum, plasma, or erythrocyte membrane odd chain fatty acids.

In some embodiments, the methods provided herein increase levels ofserum, plasma, or erythrocyte membrane very long even chain fatty acids.In further embodiments, levels of serum, plasma, or erythrocyte membranevery long even chain fatty acids may increase following administrationof one or more odd chain fatty acids, or a salt or derivative thereof.

In some embodiments, the methods provided herein modulate markers of ananemic condition when the markers provide a clinical indication.

In some embodiments, the methods provided herein alleviate symptoms ofan anemic condition.

In some embodiments, the methods provided herein reduce risk of anemia.

In some embodiments, the condition treated is anemia of chronic disease.

In some embodiments, the methods provided herein increase levels ofserum very long even chain fatty acids.

Provided herein are methods for treating including the step ofadministering a dose of a fatty acid, such as an odd chain fatty acid ora very long even chain fatty acid, at a predetermined interval, or at aninterval left to the discretion of the subject.

In some embodiments, the compounds and methods provided herein mayprovide a threshold serum, plasma, or red blood cell membrane percentageof an odd chain fatty acid relative to all serum, plasma, or red bloodcell membrane fatty acids, respectively. For example, the thresholdvalue may be a value of about 0.05% or lower to 90% or higher, e.g., avalue of at least about 0.05%, at least about 0.1%, at least about 0.2%,at least about 0.3%, at least about 0.4%, at least about 0.5%, at leastabout 0.6%, at least about 0.7%, at least about 0.8%, at least about0.9%, at least about 1.0%, at least about 1.1%, at least about 1.2%, atleast about 1.3%, at least about 1.4%, at least about 1.5%, at leastabout 1.6%, at least about 1.7%, at least about 1.8%, at least about1.9%, at least about 2.1%, at least about 2.2%, at least about 2.3%, atleast about 2.4%, at least about 2.5%, at least about 2.6%, at leastabout 2.7%, at least about 2.8%, at least about 2.9%, at least about3.0%, at least about 3.5%, at least about 4.0%, at least about 4.5%, atleast about 5%, at least about 6%, at least about 7%, at least about 8%,at least about 9%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,or more than 90%.

In some embodiments, the compounds and methods provided herein mayprovide an increase above a baseline value (e.g., pretreatment value ina patient being treated, or general value observed in a particularpatient population) in a serum or plasma concentration of an odd chainfatty acid, or red blood cell membrane concentration of an odd chainfatty acid. For example, a serum or plasma odd chain fatty acid or redblood cell membrane concentration of an odd chain fatty acid may beincreased by at least about 1 μg/ml, at least about 2 μg/ml, at leastabout 3 μg/ml, at least about 4 μg/ml, at least about 5 μg/ml, at leastabout 6 μg/ml, at least about 7 μg/ml, at least about 8 μg/ml, at leastabout 9 μg/ml, at least about 10 μg/ml, at least about 15 μg/ml, atleast about 20 μg/ml, at least about 25 μg/ml, at least about 30 μg/ml,at least about 35 μg/ml, at least about 40 μg/ml, at least about 45μg/ml, at least about 50 μg/ml, or more than 50 μg/ml. In someembodiments, the serum concentration of an odd chain fatty acid, or redblood cell membrane concentration of an odd chain fatty acid mayincrease above a baseline value (e.g., pretreatment value in a patientbeing treated, or general value observed in a particular patientpopulation) by at least about 0.01×10⁻⁴ M, at least about 0.05×10⁻⁴ M,at least about 0.1×10⁻⁴ M, at least about 0.2×10⁻⁴ M, at least about0.3×10⁻⁴ M, at least about 0.4×10⁻⁴ M, at least about 0.5×10⁻⁴ M, atleast about 0.6×10⁻⁴ M, at least about 0.7×10⁻⁴ M, at least about0.8×10⁻⁴ M, at least about 0.9×10⁻⁴ M, at least about 1×10⁻⁴ M, at leastabout 2×10⁻⁴ M, or at least about 3×10⁻⁴ M.

In some embodiments, the compounds and methods provided herein mayprovide an increase in serum or plasma total odd chain fatty acids, orred blood cell membrane total odd chain fatty acids. For example, serumtotal odd chain fatty acids, or red blood cell membrane total odd chainfatty acids, may be increased above a baseline value (e.g., pretreatmentvalue in a patient being treated, or general value observed in aparticular patient population) by at least about 5 μg/ml, at least about6 μg/ml, at least about 7 μg/ml, at least about 8 μg/ml, at least about9 μg/ml, at least about 10 μg/ml, at least about 15 μg/ml, at leastabout 20 μg/ml, at least about 25 μg/ml, at least about 30 μg/ml, atleast about 35 μg/ml, at least about 40 μg/ml, at least about 45 μg/ml,at least about 50 μg/ml, at least about 60 μg/ml, at least about 70μg/ml, at least about 80 μg/ml, at least about 90 μg/ml, at least about100 μg/ml, at least about 150 μg/ml, at least about 200 μg/ml, at leastabout 250 μg/ml, at least about 300 μg/ml, at least about 350 μg/ml, atleast about 400 μg/ml, at least about 450 μg/ml, at least about 500μg/ml, or more than 500 μg/ml.

In some embodiments, the compounds and methods provided herein mayprovide an increase above a baseline value (e.g., pretreatment value ina patient being treated, or general value observed in a particularpatient population) in a serum, plasma, or red blood cell membrane oddchain fatty acids relative to all serum or red blood cell membrane fattyacids, respectively. For example, a serum, plasma, or red blood cellmembrane odd chain fatty acid may be increased above a baseline value(e.g., pretreatment value in a patient being treated, or general valueobserved in a particular patient population) by at least about 0.01%, atleast about 0.05%, at least about 0.1%, at least about 0.2%, at leastabout 0.3%, at least about 0.4%, at least about 0.5%, at least about0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, atleast about 1%, at least about 1.1%, at least about 1.2%, at least about1.3%, at least about 1.4%, at least about 1.5%, at least about 1.6%, atleast about 1.7%, at least about 1.8%, at least about 1.9%, at leastabout 2%, at least about 2.1%, at least about 2.2%, at least about 2.3%,at least about 2.4%, at least about 2.5%, at least about 2.6%, at leastabout 2.7%, at least about 2.8%, at least about 2.9%, at least about 3%,at least about 3.5%, at least about 4%, at least about 4.5%, at leastabout 5%, or more than 5%.

In some embodiments, an odd chain fatty acid is administered to maintainserum or plasma total percent of the odd chain fatty acid, or all oddchain fatty acids, above a predetermined threshold value. In variationsof these embodiments, the odd chain fatty acid is heptadecanoic acid. Infurther variations, the odd chain fatty acid is administered to maintainserum phospholipid percent of the odd chain fatty acid, or all odd chainfatty acids, above about 0.1%, about 0.2%, about 0.3%, about 0.4%, about0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about1.2%, about 1.4%, about 1.6%, about 1.8%, about 2%, about 2.2%, about2.4%, or about 2.6%.

In some embodiments, a composition or method provided herein may providean increase in hematocrit levels. For example, a hematocrit level may beincreased above a baseline value (e.g., pretreatment value in a patientbeing treated, or general value observed in a particular patientpopulation) by at least about 1%, at least about 2%, at least about 3%,at least about 5%, at least about 7%, at least about 10%, at least about15%, at least about 20%, at least about 25%, or more than 25%.

In some embodiments, a composition or method provided herein may providean increase in hematocrit level to provide a specified value. Forexample, a hematocrit level may be increased to at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, or at least about 50%.

In some embodiments, a composition or method provided herein may providean increase in hemoglobin level. For example, a hemoglobin level may beincreased above a baseline value (e.g., pretreatment value in a patientbeing treated, or general value observed in a particular patientpopulation) by at least about 0.1 g/dL, at least about 0.2 g/dL, atleast about 0.5 g/dL, at least about 1 g/dL, at least about 1.5 g/dL, atleast about 2 g/dL, at least about 2.5 g/dL, or at least about 3 g/dL.

In some embodiments, the compositions and methods provided hereinmodulate a marker of anemia. In certain embodiments, the marker isserum, plasma, or red blood cell membrane very long even chain fattyacid percentage; serum, plasma, or red blood cell membrane concentrationof a very long even chain fatty acid; serum, plasma, or red blood cellmembrane total very long even chain fatty acid, serum ferritin, serumiron, transferrin saturation, red cell count, reticulocytes, hematocritor packed cell volume, red blood cell distribution width, meancorpuscular hemoglobin concentration (MCHC), mean corpuscular volume(MCV), mean corpuscular hemoglobin (MCH), hemoglobin, white cell count,iron deficiency, serum iron, total iron-binding capacity, transferrinsaturation, zinc protoporphyrin, or serum sTfRs. In some embodiments,the very long even chain fatty acid is measured as a constituent ofglycolipids. In further embodiments, the very long even chain fatty acidis measured as a constituent of a type of lipid, for example, aphospholipid, a sphingolipid, a membrane lipid, a glycolipid, or atriglyceride.

In some embodiments, the methods provided herein include the step ofmeasuring the concentration of a marker of anemia. One of skill in theart will be able to perform suitable methods for such measurements,including but not limited to those described herein.

In some embodiments, the compounds and methods provided herein mayprovide a threshold serum, plasma, or red blood cell membrane percentageof a very long even chain fatty acid relative to all serum or red bloodcell membrane fatty acids, respectively. For example, the thresholdvalue may be a value of about 0.05% or lower to 90% or higher, e.g., avalue of at least about 0.05%, at least about 0.1%, at least about 0.2%,at least about 0.3%, at least about 0.4%, at least about 0.5%, at leastabout 0.6%, at least about 0.7%, at least about 0.8%, at least about0.9%, at least about 1.0%, at least about 1.1%, at least about 1.2%, atleast about 1.3%, at least about 1.4%, at least about 1.5%, at leastabout 1.6%, at least about 1.7%, at least about 1.8%, at least about1.9%, at least about 2.1%, at least about 2.2%, at least about 2.3%, atleast about 2.4%, at least about 2.5%, at least about 2.6%, at leastabout 2.7%, at least about 2.8%, at least about 2.9%, at least about3.0%, at least about 3.5%, at least about 4.0%, at least about 4.5%, atleast about 5%, at least about 6%, at least about 7%, at least about 8%,at least about 9%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,or more than 90%.

In some embodiments, the compounds and methods provided herein mayprovide an increase above a baseline value (e.g., pretreatment value ina patient being treated, or general value observed in a particularpatient population) in a serum or plasma concentration of a very longeven chain fatty acid, or red blood cell membrane concentration of avery long even chain fatty acid. For example, a serum very long evenchain fatty acid or red blood cell membrane concentration of a very longeven chain fatty acid may be increased by at least about 0.01 μg/ml, atleast about 0.05 μg/ml, at least about 0.1 μg/ml, at least about 0.4μg/ml, 1 μg/ml, at least about 2 μg/ml, at least about 3 μg/ml, at leastabout 4 μg/ml, at least about 5 μg/ml, at least about 6 μg/ml, at leastabout 7 μg/ml, at least about 8 μg/ml, at least about 9 μg/ml, at leastabout 10 μg/ml, at least about 15 μg/ml, at least about 20 μg/ml, atleast about 25 μg/ml, at least about 30 μg/ml, at least about 35 μg/ml,at least about 40 μg/ml, at least about 45 μg/ml, at least about 50μg/ml, or more than 50 μg/ml. In some embodiments, the serumconcentration of a very long even chain fatty acid, or red blood cellmembrane concentration of a very long even chain fatty acid may increaseabove a baseline value (e.g., pretreatment value in a patient beingtreated, or general value observed in a particular patient population)by at least about 0.001×10⁻⁴ M, at least about 0.005×10⁻⁴ M, at leastabout 0.05×10⁻⁴ M, at least about 0.01×10⁻⁴ M, at least about 0.05×10⁻⁴M, at least about 0.1×10⁻⁴ M, at least about 0.2×10⁻⁴ M, at least about0.3×10⁻⁴ M, at least about 0.4×10⁻⁴ M, at least about 0.5×10⁻⁴ M, atleast about 0.6×10⁻⁴ M, at least about 0.7×10⁻⁴ M, at least about0.8×10⁻⁴ M, at least about 0.9×10⁻⁴ M, at least about 1×10⁻⁴ M, at leastabout 2×10⁻⁴ M, or at least about 3×10⁻⁴ M.

In some embodiments, the compounds and methods provided herein mayprovide an increase in serum or plasma total very long even chain fattyacids, or red blood cell membrane total very long even chain fattyacids. For example, serum total very long even chain fatty acids, or redblood cell membrane total very long even chain fatty acids, may beincreased above a baseline value (e.g., pretreatment value in a patientbeing treated, or general value observed in a particular patientpopulation) by at least about 0.05 μg/ml, at least about 0.1 μg/ml, atleast about 0.5 μg/ml, at least about 1 μg/ml, at least about 5 μg/ml,at least about 6 μg/ml, at least about 7 μg/ml, at least about 8 μg/ml,at least about 9 μg/ml, at least about 10 μg/ml, at least about 15μg/ml, at least about 20 μg/ml, at least about 25 μg/ml, at least about30 μg/ml, at least about 35 μg/ml, at least about 40 μg/ml, at leastabout 45 μg/ml, at least about 50 μg/ml, at least about 60 μg/ml, atleast about 70 μg/ml, at least about 80 μg/ml, at least about 90 μg/ml,at least about 100 μg/ml, at least about 150 μg/ml, at least about 200μg/ml, at least about 250 μg/ml, at least about 300 μg/ml, at leastabout 350 μg/ml, at least about 400 μg/ml, at least about 450 μg/ml, atleast about 500 μg/ml, or more than 500 μg/ml.

In some embodiments, the compounds and methods provided herein mayprovide an increase above a baseline value (e.g., pretreatment value ina patient being treated, or general value observed in a particularpatient population) in a serum, plasma or red blood cell membrane verylong even chain fatty acids relative to all serum, plasma or red bloodcell membrane fatty acids, respectively. For example, a serum or redblood cell membrane very long even chain fatty acid may be increasedabove a baseline value (e.g., pretreatment value in a patient beingtreated, or general value observed in a particular patient population)by at least about 0.01%, at least about 0.05%, at least about 0.1%, atleast about 0.2%, at least about 0.3%, at least about 0.4%, at leastabout 0.5%, at least about 0.6%, at least about 0.7%, at least about0.8%, at least about 0.9%, at least about 1%, at least about 1.1%, atleast about 1.2%, at least about 1.3%, at least about 1.4%, at leastabout 1.5%, at least about 1.6%, at least about 1.7%, at least about1.8%, at least about 1.9%, at least about 2%, at least about 2.1%, atleast about 2.2%, at least about 2.3%, at least about 2.4%, at leastabout 2.5%, at least about 2.6%, at least about 2.7%, at least about2.8%, at least about 2.9%, at least about 3%, at least about 3.5%, atleast about 4%, at least about 4.5%, at least about 5%, or more than 5%.

In some embodiments, a composition or method provided herein may providean increase in hematocrit levels. For example, a hematocrit level may beincreased above a baseline value (e.g., pretreatment value in a patientbeing treated, or general value observed in a particular patientpopulation) by at least about 1%, at least about 2%, at least about 3%,at least about 5%, at least about 7%, at least about 10%, at least about15%, at least about 20%, at least about 25%, or more than 25%.

In some embodiments, a very long even chain fatty acid is administeredto maintain serum or plasma phospholipid percent of the very long evenchain fatty acid, or all very long even chain fatty acids, above apredetermined threshold value. In variations of these embodiments, thevery long even chain fatty acid is behenic acid. In further variations,the very long even chain fatty acid is administered to maintain serumphospholipid percent of the very long even chain fatty acid, or all verylong even chain fatty acids, above about 0.1%, about 0.2%, about 0.3%,about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%,about 1%, about 1.2%, about 1.4%, about 1.6%, about 1.8%, about 2%,about 2.2%, about 2.4%, or about 2.6%.

In some embodiments, a composition or method provided herein may providean increase in packed cell volume. For example, a packed cell volumelevel may be increased above a baseline value (e.g., pretreatment valuein a patient being treated, or general value observed in a particularpatient population) by at least about 1%, at least about 2%, at leastabout 3%, at least about 5%, at least about 7%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, or more than25%.

In some embodiments, a composition or method provided herein may providean increase in red blood cell count. For example, a red blood cell countlevel may be increased above a baseline value (e.g., pretreatment valuein a patient being treated, or general value observed in a particularpatient population) by at least about 0.1 cells/μL, at least about 0.2cells/μL, at least about 0.3 cells/μL, at least about 0.4 cells/μL, atleast about 0.5 cells/L, at least about 0.6 cells/L, at least about 0.7cells/L, at least about 0.8 cells/μL, at least about 0.9 cells/L, atleast about 1 cell/μL, at least about 1.2 cells/L, at least about 1.4cells/L, at least about 1.6 cells/L, or at least about 2 cells/L.

In some embodiments, a composition or method provided herein may providean increase in hemoglobin levels. For example, a hemoglobin level may beincreased above a baseline value (e.g., pretreatment value in a patientbeing treated, or general value observed in a particular patientpopulation) by at least about 1 g/dL, at least about 2 g/dL, at leastabout 3 g/dL, at least about 4 g/dL, at least about 5 g/dL, at leastabout 6 g/dL, at least about 7 g/dL, at least about 8 g/dL, at leastabout 9 g/dL, or at least about 10 g/dL.

In some embodiments, a composition or method provided herein may providean increase in MCV levels. For example, a MCV level may be increasedabove a baseline value (e.g., pretreatment value in a patient beingtreated, or general value observed in a particular patient population)by at least about 5 fL, at least about 10 fL, at least about 15 fL, atleast about 20 fL, at least about 25 fL, at least about 30 fL, at leastabout 35 fL, at least about 40 fL, or at least about 50 fL.

In some embodiments, a composition or method provided herein may providean increase in MCH levels. For example, a MCH level may be increasedabove a baseline value (e.g., pretreatment value in a patient beingtreated, or general value observed in a particular patient population)by at least about 1%, at least about 2%, at least about 3%, at leastabout 5%, at least about 7%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, or more than 25%.

In some embodiments, a composition or method provided herein may providean increase in MCHC levels. For example, a MCHC level may be increasedabove a baseline value (e.g., pretreatment value in a patient beingtreated, or general value observed in a particular patient population)by at least about 0.01 g/dL, at least about 0.05 g/dL, at least about0.1 g/dL, at least about 0.5 g/dL, at least about 1 g/dL, at least about2 g/dL, at least about 3 g/dL, at least about 4 g/dL, at least about 5g/dL, at least about 7 g/dL, or at least about 10 g/dL.

In some embodiments, a composition or method provided herein may providea reduction in RBC distribution width. For example, a RBC distributionwidth level may be reduced below a baseline value (e.g., pretreatmentvalue in a patient being treated, or general value observed in aparticular patient population) by at least about 0.05%, at least about0.1%, at least about 0.5%, at least about 1%, at least about 2%, atleast about 3%, at least about 5%, at least about 7%, at least about10%, at least about 15%, at least about 20%, at least about 25%, or morethan 25%.

Combination Therapies

In some embodiments, the compounds disclosed herein, such as an oddchain fatty acid, or a salt or derivative thereof, or a very long evenchain fatty acid, or a salt or derivative thereof, or a pharmaceuticalcomposition that includes a compound described herein, or a salt orderivative thereof, may be used in combination with one or moreadditional active agents. Examples of additional active agents that canbe used in combination with a compound of an odd chain fatty acid or avery long even chain fatty acid, or a salt or derivative thereof, or acomposition that includes a compound of an odd chain fatty acid, or asalt or derivative thereof, a very long even chain fatty acid, or a saltor derivative thereof, include, but are not limited to, agents currentlyused for treating conditions provided herein, and as otherwise known tomedical science.

In some embodiments, a compound of an odd chain fatty acid, or a salt orderivative thereof, a very long even chain fatty acid, or a salt orderivative thereof, or a composition that includes a compound of an oddchain fatty acid, or a salt or derivative thereof, a very long evenchain fatty acid, or a salt or derivative thereof, can be used with one,two, three or more additional active agents described herein. Suchagents include, but are not limited to, a second fatty acid, such as anodd chain fatty acid or a very long even chain fatty acid, or a salt orderivative thereof. In some embodiments, a composition can include atleast one odd chain fatty acid, or a salt or derivative thereof, and atleast one very long even chain fatty acid, or a salt or derivativethereof.

In some embodiments, a compound of an odd chain fatty acid, or a salt orderivative thereof, a very long even chain fatty acid, or a salt orderivative thereof, or a composition that includes a compound of an oddchain fatty acid, or a salt or derivative thereof, a very long evenchain fatty acid, or a salt or derivative thereof, can be used (forexample, administered or ingested) in combination with another agent oragents for treatment, prevention, maintenance, or prophylaxis of ananemic condition selected from hemolytic anemias (including but notlimited to thalassemias, hereditary spherocytosis, hereditaryelliptocytosis, glucose-6-phosphate dehydrogenase deficiency, pyruvatekinase deficiency, immune hemolytic anemia, alloimmune hemolytic anemia,drug-induced hemolytic anemia, mechanical hemolytic anemias, andparoxysmal nocturnal hemoglobinuria), anemia of chronic disease (whereinthe underlying condition can be, for example, autoimmune disorders (suchas, for example Crohn disease, systemic lupus erythematosus, rheumatoidarthritis, type 1 diabetes, and ulcerative colitis), neoplasticdisorders including cancer (such as, for example lymphoma and Hodgkindisease), long-term infections (such as, for example bacterial, viral,and fungal infections), rheumatoid arthritis, ulcerative colitis,Hodgkin disease, metabolic syndrome, diabetes (for example, type 2diabetes), and other causes of chronic inflammation), anemia, aplasticanemias (including but not limited to congenital hypoplastic anemia,Diamond-Blackfan anemia and Fanconi anemia), iron deficiency anemia,anemias of abnormal RBC size (including but not limited to megaloblasticanemia and microcytic anemia), vitamin deficiency anemias (including butnot limited to pernicious anemia) anemia of RBC mutation (including butnot limited to thalassemia, sideroblastic anemia and sickle cellanemia). For example, a compound of a fatty acid, such as an odd chainfatty acid or a very long even chain fatty acid, disclosed herein can beused in combination with one or more agents selected from ironchelators, albiglutide, aleglitazar, balaglitazone, canagliflozin,CJ-30001 (CJ Cheiljedang Corporation), CJ-30002 (CJ CheiljedangCorporation), Diamyd® (glutamic acid decarboxylase (rhGAD65)),dulaglutide, exendin 4, gemigliptin, lixisenatide, lobeglitazone,shengke I (Tibet Pharmaceuticals), SK-0403 (Sanwa Kagaku Kenkyusho),teneligliptin, teplizumab, tofogliflozin, acarbose, alogliptin benzoate,chlorpropamide, Diab II (Biotech Holdings), exenatide, glibenclamide,gliclazide, glimepiride, glipizide, gliquidone, glisentide, glisolamide,HL-002 (HanAII Biopharma), insulin (human), insulin, insulin analogue(Eli Lilly®), insulin aspart, insulin detemir, insulin glargine, insulinlispro, Janumet®, linagliptin, liraglutide, metformin, miglitol,mitiglinide, nateglinide, Novo Mix 30® (Novo Nordisk®) pioglitazone,pramlintide, repaglinide, rosiglitazone maleate, saxagliptin,sitagliptin, Tresiba, tolazamide, tolbutamide, vildagliptin, voglibose,bezafibrate, diflunisal, cinnamic acid, carbutamide, glyburide(glibenclamide), glibomuride, glyhexamide, phenbutamide, andtolcyclamide or with one or more agents selected from a class of agents,where the classes include sulfonylureas, non-sulfonylurea secretagogues,glucagon-like peptides, exendin-4 polypeptides, beta 3 adrenoceptoragonists, PPAR agonists, dipeptidyl peptidase IV inhibitors, biguanides,alpha-glucosidase inhibitors, immunomodulators, statins andstatin-containing combinations, angiotensin converting enzymeinhibitors, adeno sine A1 receptor agonists, adenosine A2 receptoragonists, aldosterone antagonists, alpha 1 adrenoceptor antagonists,alpha 2 adrenoceptor agonists, alpha 2 adrenoceptor agonists,angiotensin receptor antagonists, antioxidants, ATPase inhibitors,atrial peptide agonists, beta adrenoceptor antagonists, calcium channelagonists, calcium channel antagonists, diguanides, diuretics, dopamineD1 receptor agonists, endopeptidase inhibitors, endothelin receptorantagonists, guanylate cyclase stimulants, phosphodiderivativease Vinhibitors, protein kinase inhibitors, Cdc2 kinase inhibitors, renininhibitors, thromboxane synthase inhibitors, vasopeptidase inhibitors,vasopressin I antagonists, vasopressin 2 antagonists, angiogenesisinhibitors, advanced glycation end product inhibitors, bile acid bindingagents, bile acid transport inhibitors, bone formation stimulants,apolipoprotein A1 agonists, DNA topoisomerase inhibitors, cholesterolabsorption inhibitors, cholesterol antagonists, cholderivativeylderivative transfer protein antagonists, cytokine synthesis inhibitors,DNA polymerase inhibitors, dopamine D2 receptor agonists, endothelinreceptor antagonists, growth hormone antagonists, insulin sensitizers,lipase inhibitors, lipid peroxidation inhibitors, lipoprotein Aantagonists, microsomal transport protein inhibitors, microsomaltriglyceride transfer protein inhibitors, nitric oxide synthaseinhibitors, oxidizing agents, phospholipase A2 inhibitors, radicalformation agonists, platelet aggregation antagonists, prostaglandinsynthase stimulants, reverse cholesterol transport activators, rhokinase inhibitors, selective estrogen receptor modulators, squaleneepoxidase inhibitors, squalene synthase inhibitors, thromboxane A2antagonists, amylin agonists, cannabinoid receptor antagonists,cholecystokinin A agonists, corticotropin-releasing factor agonists,dopamine uptake inhibitors, G protein-coupled receptor modulators,glutamate antagonists, glucagon-like peptide-1 agonists lipaseinhibitors, melanin-concentrating hormone receptor antagonists, nervegrowth factor agonists, neuropeptide Y agonists, neuropeptide Yantagonists, SNRIs, protein tyrosine phosphatase inhibitors, serotonin2C receptor agonists, or with other agents such as central nervoussystem agents that affect neurotransmitters or neural ion channels,including antidepressants (bupropion), noradrenalin reuptake inhibitors(GW320659), selective serotonin 2c receptor agonists, selective κHT 2creceptor agonists, antiseizure agents (topiramate, zonisamide), dopamineantagonists, cannabinoid-1 receptor antagonists (CB-1 receptorantagonists) (rimonabant); leptin/insulin/central nervous system pathwayagents, including leptin analogues, leptin transport and/or leptinreceptor promoters, ciliary neurotrophic factor (Axokine), neuropeptideY and agouti-related peptide antagonists, pro-opiomelanocortin andcocaine and amphetamine regulated transcript promoters,α-melanocyte-stimulating hormone analogues, melanocoritin-4 receptoragonists, and agents that affect insulin metabolism/activity, whichinclude protein-tyrosine phosphatase-IB inhibitors, peroxisomeproliferator activated receptor-.gamma. receptor antagonists,short-acting bromocriptine (ergoset), somatostatin agonists(octreotide), and adiponectin/Acrp30 (Famoxin or Fatty Acid MetabolicOxidation Inducer); gastrointestinal-neural pathway agents, includingthose that increase cholecystokinin activity (CCK), PYY activity, NPYactivity, and PP activity, increase glucagon-like peptide-1 activity(exendin 4, dipeptidyl peptidase IV inhibitors), and those that decreaseghrelin activity, as well as amylin analogues (pramlintide); agents thatmay increase resting metabolic rate (selective β-3 stimulators/agonist,uncoupling protein homologues, and thyroid receptor agonists); othermore diverse agents, including melanin concentrating hormoneantagonists, phytostanol analogues, functional oils, P57, amylaseinhibitors, growth hormone fragments, synthetic analogues ofdehydroepiandrosterone sulfate, antagonists of adipocyte11B-hydroxysteroid dehydrogenase type 1 activity,corticotropin-releasing hormone agonists, inhibitors of fatty acidsynthesis (cerulenin and C75), carboxypeptidase inhibitors,indanone/indanols, aminosterols (trodusquemine/trodulamine), and othergastrointestinal lipase inhibitors (ATL962); amphetamines, such asdextroamphetamine; other sympathomimetic adrenergic agents, includingphentermine, benzphetamine, phendimetrazine, mazindol, anddiethylpropion; or with one or more agents selected from ecopipam;oxyntomodulin (OM); inhibitors of glucose-dependent insulinotropicpolypeptide (GIP); gastrin-releasing peptide; neuromedin B;enterostatin; amfebutamone, SR-58611; CP-045598; AOD-0604; QC-BT16;rGLP-1; 1426 (HMR-1426); N-5984; ISIS-1 13715; solabegron; SR-147778;Org-34517; melanotan-II; cetilistat; c-2735; c-5093; c-2624; APD-356;radafaxine; fluasterone; GP-389255; 856464; S-2367; AVE-1625; T-71;oleoyl-estrone; peptide YY [3-36] intranasal; androgen receptoragonists; PYY 3-36; DOV-102677; tagatose; SLV-319; 1954 (Aventis PharmaAG); oxyntomodulin, Thiakis; bromocriptine, PLIVA;diabetes/hyperlipidemia therapy, Yissum; CKD-502; thyroid receptor betaagonists; beta-3 adrenoceptor agonist; CDK-A agonists; galaninantagonist; dopamine D1 D2 agonists; melanocortin modulators;verongamine; neuropeptide Y antagonists; melanin-concentrating hormonereceptor antagonists; dual PPAR alpha/gamma agonists; CGEN-P-4; kinaseinhibitors; human MCH receptor antagonists; GHS-R antagonists; ghrelinreceptor agonists; DG70 inhibitors; cotinine; CRF-BP inhibitors;urocortin agonists; UCL-2000; impentamine; 0-3 adrenergic receptor;pentapeptide MC4 agonists; trodusquemine; GT-2016; C-75; CPOP; MCH-1receptor antagonists; RED-103004; aminosterols; orexin-1 antagonists;neuropeptide Y5 receptor antagonists; DRF-4158; PT-15; PTPaseinhibitors; A37215; SA-0204; glycolipid metabolites; MC-4 agonist;produlestan; PTP-1B inhibitors; GT-2394; neuropeptide Y5 antagonists;melanocortin receptor modulators; MLN-4760; PPAR gamma/delta dualagonists; NPY5RA-972; 5-HT2C receptor agonist; neuropeptide Y5 receptorantagonists (phenyl urea analogs); AGRP/MC4 antagonists; neuropeptide Y5antagonists (benzimidazole); glucocorticoid antagonists; MCHR1antagonists; Acetyl-CoA carboxylase inhibitors; R-1496; HOB 1modulators; NOX-B11; peptide YY 3-36 (eligen); 5-HT 1 modulators;pancreatic lipase inhibitors; GRC-1087; CB-1 antagonists; MCH-1antagonists; LY-448100; bombesin BRS3 agonists; ghrelin antagonists; MC4antagonists; stearoyl-CoA desaturase modulators; PPAR pan agonists;EP-01492; hormone-sensitive lipase inhibitors; fatty acid-bindingprotein 4 inhibitors; thiolactone derivatives; protein tyrosinephosphatase IB inhibitors; MCH-1 antagonist; P-64; PPAR gamma ligands;melanin concentrating hormone antagonists; thiazole gastroprokinetics;PA-452; T-226296; A-331440; immunodrug vaccines; diabetes/obesitytherapeutics (Bioagency, Biofrontera Discovery GmbH); P-7 (Genfit);DT-011 M; PTP1B inhibitor; anti-diabetic peptide conjugates; KATPagonists; obesity therapeutics (Lexicon); 5-HT2 agonists; MCH-1 receptorantagonists; GMAD-1/GMAD-2; STG-a-MD; angiogenesis inhibitors; Gprotein-coupled receptor agonists; nicotinic therapeutics (ChemGenex);anti-obesity agents (Abbott); melanin concentrating hormone; GW-594884A;MC-4R agonist; histamine H3 antagonists; orphan GPCR modulators;MITO-3108; NLC-002; HE-2300; IGF/BBP-2-13; 5-HT2C agonists; ML-22952;neuropeptide Y receptor antagonists; AZ-40140; anti-obesity therapy(Nisshin Flour); GNTI; melanocortin receptor modulators; alpha-amylaseinhibitors; beta-3 adrenoceptor agonists; ob gene products (Eli Lilly &Co.); SWR-0342-SA; SWR-0335; SP-18904; oral insulin mimetics; obesitytherapeutics (7TM Pharma); beta-hydroxysteroid dehydrogenase (HSD)inhibitors; QRX-431; E-6776; RI-450; melanocortin-4 antagonists;melanocortin 4 receptor agonists; obesity therapeutics (CuraGen); leptinmimetics; A-74498; second-generation leptin; NBI-103; CL-314698;CP-114271; beta-3 adrenoceptor agonists; NMI-8739; UCL-1283; BMS-192548;CP-94253; PD-160170; nicotinic agonist; LG-100754; SB-226552; LY-355124;CKD-711; L-751250; PPAR inhibitors; G-protein therapeutics; obesitytherapy (Amylin Pharmaceuticals Inc.); BW-1229; monoclonal antibody(ObeSys/CAT); L-742791; (S)-sibutramine; MBU-23; YM-268; BTS-78050;tubby-like protein genes; genomics (eating disorders; Allelix/Lilly);MS-706; GI-264879A; GW-409890; FR-79620 analogs; obesity therapy(Hybrigenics SA); ICI-198157; ESP-A; 5-HT2C agonists; PD-170292;AIT-202; LG-100641; GI-181771; anti-obesity therapeutics (Genzyme);leptin modulator; GHRH mimetics; obesity therapy (YamanouchiPharmaceutical Co. Ltd.); SB-251023; CP-331684; BIBO-3304;cholesten-3-ones; LY-362884; BRL-48962; PY-1 antagonists; A-71378;®-didesmethylsibutramine; obesity therapeutics (Bristol-Myers SquibbCo.); obesity therapeutics (Ligand Pharmaceuticals Inc.); LY-226936; NPYantagonists; CCK-A agonists; FPL-14294; PD-145942; ZA-7114; CL-316243;SR-58878; R-1065; BDBP-3226; HP-228; talibegron; FR-165914; AZM-008;AZM-016; AZM-120; AZM-090; AZM-131; AZM-132; AZM-134; AZM-127; AZM-083;AZM-115; AZM-140; vomeropherin; BMS-187257; D-3800; gene discovery(Axys/Glaxo); BRL-26830A; SX-013; ERR modulators; adipsin; AC-253;A-71623; A-68552; BMS-210285; TAK-677; MPV-1743; obesity therapeutics(Modex); GI-248573; exopipam; SSR-125180; obesity therapeutics (MelacureTherapeutics AB); BRL-35135; SR-146131; P-57; CGP-71583A; RF-1051;BMS-196085; manifaxine; DMNJ (Korea Research Institute of Bioscience andBiotechnology); BVT-5182; LY-255582; SNX-024; galanin antagonists;neurokinin-3 antagonists; dexfenfluramine; mazindol; diethylpropion;phendimetrazine; benzphetamine; amfebutmone; sertraline; AOD-9604;ATL-062; BVT-933; GT389-255; SLV319; HE-2500; PEG-axokine; L-796568; andABT-239; rimonabant, sibutramine, orlistat, PYY or an analog thereof,CB-1 antagonist, leptin, phentermine, and exendin analogs; GPR1 19agonists (e.g., anandamide; AR-231, 453; MBX-2982; Oleoylethanolamide;PSN-365,963; PSN-632,408; palmitoylethanolamide); GPR120 agonists; GPR40 agonists; and SGLT2 inhibitors.

Additionally, a fatty acid or salt or derivative as provided herein canbe used in combination with one or more agents selected from Altoprev(lovastatin), Crestor (rosuvastatin), Lescol (fluvastatin), Lipitor(atorvastatin), Livalo (pitavastatin), Pravachol (pravastatin), Zocor(simvastatin), an anti-platelet medication, a beta blocker, an ACEinhibitor, a calcium channel blocker, a diuretic, anticoagulants,aspirin, bile acid sequestrants, Ezetimibe, Fibrates, GlycoproteinIIb/IIIa Receptor Inhibitors, Niacin (Nicotinic Acid), Nitrates,Platelet Inhibitors, Thrombolytics, lisinopril oral, atenolol oral,Bystolic oral, Diovan oral, hydrochlorothiazide oral, metoprololsuccinate oral, amlodipine oral, Norvasc oral, Toprol XL oral, Benicaroral, metoprolol tartrate oral, losartan oral,lisinopril-hydrochlorothiazide oral, clonidine HCl oral, Diovan HCToral, Cozaar oral, propranolol oral, spironolactone oral, Azor oral,carvedilol oral, Coreg oral, Benicar HCT oral, Exforge oral, Avaprooral, Lotrel oral, verapamil oral, furosemide oral, Lasix oral, Hyzaaroral, Tekturna oral, enalapril maleate oral, Micardis oral,losartan-hydrochlorothiazide oral, ramipril oral, Lopressor oral, Altaceoral, Micardis HCT oral, Avalide oral, diltiazem oral,triamterene-hydrochlorothiazide oral, labetalol oral, terazosin oral,amlodipine-benazepril oral, hydralazine oral, Atacand oral, benazepriloral, Tribenzor oral, triamterene oral, doxazosin oral, nifedipine oral,Ziac oral, Aldactone oral, Maxzide oral, Cartia XT oral, prazosin oral,Cardizem CD oral, Zestril oral, Dyazide oral, bisoprolol fumarate oral,Tenex oral, Tenormin oral, Coreg CR oral, Prinivil oral, valsartan oral,atenolol-chlorthalidone oral, Edarbyclor oral,benazepril-hydrochlorothiazide oral, ferrous sulfate oral, Ferrlecitintravenous, Feraheme intravenous, Feosol oral, Infed injection, Integraoral, Ferrex 150 Forte oral, Tandem Dual Action oral, Ferrex 150 oral,ferrous gluconate oral, Corvite 150 oral, Integra F oral, NovaFerrumoral, Iron (ferrous sulfate) oral, Vitron-C oral, Folic acid,corticosteroids, rituximab, IVIG, prednisone, methylprednisolone oral,Kenalog injection, Medrol (Pak) oral, Medrol oral, dexamethasone oral,Depo-Medrol injection, prednisolone oral, DexPak 13 Day oral,Solu-Medrol intravenous, hydrocortisone oral, Cortef oral, Deltasoneoral, triamcinolone acetonide injection, cortisone oral, cholinesteraseinhibitors such as Donepezil (Aricept), Rivastigmine (Exelon), andGalantamine (Razadyne), Memantine, Aricept, Namenda, Namenda XR,Razadyne ER, Alpha E, vitamin E, Hydergine, Namzaric, Dopamine Agonistssuch as pramipexole (Mirapex), ropinirole (Requip), rotigotine (Neupropatch) and apomorphine (Apokyn), Anticholinergics such as benztropine(Cogentin) and trihexyphenidyl, MAO-B Inhibitors such as (Eldepryl,Zelapar) and rasagiline (Azilect), COMT Inhibitors such as Entacapone(Comtan), Carbidopa/Levodopa (Sinemet®), amantadine, Tetrabenazine(Xenazine), haloperidol (Haldol), chlorpromazine, risperidone(Risperdal), quetiapine (Seroquel), olanzapine (Zyprexa), indomethacin,sulindac, etodolac, mefenamic acid, meclofenamic acid, meclofenamatesodium, flufenamic acid, tolmetin, ketorolac, diclofenac, diclofenacsodium, ibuprofen, naproxen, naproxen sodium, fenoprofen, ketoprofen,flurbiprofen, oxaprozin piroxicam, meloxicam, ampiroxicam, droxicam,lornoxicam, cinnoxicam, sudoxicam, and tenoxicam.

Additionally, a compound of a fatty acid disclosed herein can be used incombination with one or more agents selected from iron dextran, ironsumalate, polysaccharide iron, ferrus fumarate, carbonyl iron, ferrousasparto glycinate, heme iron polypeptide can be sometimes indicated,ferrus bisglycinate as can be the administration of other medicamentssuch as androgen hormones, such as erythropoietin, folic acid, vitaminB12, vitamin C, succinic acid, niacin, pyridoxine, riboflavin, biotin,thiamine, calcium formate, Aminoxin, Anadrol-50, Chromagen Forte,Epoetin alfa, Epogen, Fe C Tab Plus, FeRiva, FeRivaFA, Ferocon,Ferotrin, Ferralet 90, Ferrex 28, Ferrogels Forte, FoliTab 500,Fumatinic, Hematogen Forte, Hemetab, Integra Plus, Irospan 42/6,Lenalidomide, Maxaron Forte, Myferon 150 Forte, MyKidz Iron, NovaFerrum,Oxymetholone, Procrit, Proferrin-Forte, Pyridoxine, Repliva 21/7,Revlimid, and Tricon.

Dosing

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight, the severity of the condition,and mammalian species treated, the particular forms of the compoundsemployed, and the specific use for which these compounds are employed.The determination of effective dosage levels, that is the dosage levelsnecessary to achieve the desired result, can be accomplished by oneskilled in the art using routine methods, for example, in vivo studies.Reference may be made to, for example, “Estimating the Maximum SafeStarting Dose in Initial Clinical Trials for Therapeutics in AdultHealthy Volunteers,” U.S. Food and Drug Administration, July 2005.

In some embodiments, a method provided herein may comprise administeringa therapeutically effective amount of a composition provided herein. Insome embodiments, a therapeutically effective amount may be determinedby reference to the modulation of a marker of a condition providedherein including an anemic condition. In some embodiments, atherapeutically effective amount may be determined by reference to themodulation of a symptom of a condition provided herein. In still otherembodiments, reference may be made to established guidelines for theconditions described herein, including, but not limited to, guidelinesfor the treatment of a condition provided herein including an anemiccondition.

The dosage may vary broadly, depending upon the desired effects and thetherapeutic indication, such as marker values. Alternatively, dosagesmay be based and calculated upon the surface area or weight of thepatient, as understood by those of skill in the art. The exact dosagewill be determined on a case-by-case basis, or, in some cases, will beleft to the informed discretion of the subject. The daily dosage regimenfor an adult human patient may be, for example, an oral dose of a fattyacid, such as an odd chain fatty acid or a very long even chain fattyacid, or a salt or derivative thereof, or a mixture of a plurality offatty acids, or a salt or derivative thereof, from about 0.01 mg toabout 10000 mg, from about 1 mg to about 5000 mg, from about 5 mg toabout 2000 mg, from about 10 mg to about 1000 mg, or from about 50 mg toabout 500 mg. A single dose may include a fatty acid, or a salt orderivative thereof, in about 0.01 mg, about 0.1 mg, about 1 mg, about 5mg, about 10 mg, about 20 mg, about 50 mg, about 100 mg, about 200 mg,about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 800 mg,about 900 mg, about 1000 mg, about 2000 mg, about 5000 mg, or more. Thedosage may be adjusted according to the body mass of the subject, forexample, the dosage may be about 0.001 mg/kg, about 0.01 mg/kg, about0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg,about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8mg/kg, about 9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg,about 25 mg/kg, about 30 mg/kg, or higher. The dosage may be a singleone or a series of two or more given in the course of one or more days,as is appropriate for the individual subject. In some embodiments, thecompounds will be administered for a period of continuous therapy, forexample for about a week or more (e.g., one week, two weeks, threeweeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, ormore), for several weeks, for about a month or more (e.g., one month,two months, three months, four months, five months, six months, sevenmonths, eight months, nine months, ten months, eleven months, twelvemonths, or more), for about a year or more, or for a plurality of years.In some embodiments, a fatty acid, such as an odd chain fatty acid or avery long even chain fatty acid, or a salt or derivative thereof, can beadministered or ingested one time per day, two times per day, threetimes per day, or more.

As will be understood by those of skill in the art, in certainsituations it may be necessary to administer the compounds disclosedherein in amounts that exceed the above-stated, preferred dosage rangein order to effectively treat a subject.

Unit dosage forms can also be provided, e.g., individual packages with apremeasured amount of the composition, configured for administration ona predetermined schedule. Unit dosage forms configured foradministration one to three times a day are preferred; however, incertain embodiments it may be desirable to configure the unit dosageform for administration more than three times a day, or less than onetime per day.

Dosage amount and interval may be adjusted to the individual subject toprovide plasma levels of the active moiety which are sufficient tomaintain predetermined parameters, indicators, or marker values, orminimal effective concentration (MEC). Dosages necessary to achieve thedesired result will depend on individual characteristics and route ofadministration. However, assays, for example, HPLC assays or bioassays,may be used to determine serum concentrations.

In some embodiments, the compounds and methods provided herein may beused in conjunction with devices and methods of using devices, forexample, as provided in U.S. Pat. Nos. 7,651,845; 8,251,904; 8,251,904;4,985,015; 8,827,957; 4,252,159; 5,318,521; 4,718,430; U.S.2011/0190702; DE2615061; and in conjunction with diagnostic devices, forexample, as provided in U.S. 2012/0072236.

Diagnosis and Monitoring

Provided herein are methods for the diagnosis and monitoring ofconditions provided herein including an anemic condition.

In some embodiments, the method of diagnosis or monitoring may comprisethe step of measuring a percentage of a fatty acid, such as an odd chainfatty acid or a very long even chain fatty acid, in a bodily fluid. Insome embodiments, the method of diagnosis or monitoring may comprise thestep of measuring a marker of an anemic condition in a subject. In someembodiments, the method of diagnosis or monitoring may comprise the stepof measuring a marker of anemia of chronic disease. In some embodiments,a correlation between one marker and another may prove instructive. Insome embodiments, a condition provided herein, including an anemiccondition, may be diagnosed by reference to a threshold level of amarker of the condition, for example, serum odd chain fatty acidpercentage, serum concentration of an odd chain fatty acid, serum totalodd chain fatty acid, serum very long even chain fatty acid, serum totalvery long even chain fatty acids, or a ratio between two serum fattyacids. For example, the threshold may be determined by reference to asymptom or marker of an anemic condition.

The percentage of a fatty acid, such as an odd chain fatty acid or avery long even chain fatty acid, or a marker of an anemic condition in asubject may be monitored by any means. Samples for analysis may bederived any fluid or tissue of the subject. For example, from serum,plasma, erythrocyte membranes, urine, and feces.

Example

Dolphins at the Navy Marine Mammal Program (MMP) are a well-studieddolphin population with regard to chronic diseases and diseases ofaging, and this population is more susceptible to anemia (lower packedcell volume and lower hemoglobin) compared to wild dolphins living inSarasota Bay, Fla. (Table 1). MMP dolphins also have lower total serumodd chain saturated fatty acids and lower very long even chain saturatedfatty acids compared to Sarasota Bay dolphins (Table 1, See alsoVenn-Watson et al. (2016) Increased dietary intake of saturated fattyacid heptadecanoic acid (C17:0) associated with decreasing ferritin andalleviated metabolic syndrome in dolphins. PLOS ONE 10(7):e0132117).Proposed risk factors for anemia in MMP dolphins can include advancedage, chronic disease, and diet (see Venn-Watson S, Smith C R, Gomez F,Jensen E D (2011) Physiology of aging among healthy, older bottlenosedolphins (Tursiops truncatus): comparisons with aging humans. J CompPhys B 181:667-680). It can be hypothesized that differences in dietaryfish (and differences in certain fatty acids associated with particulartypes of fish) can be responsible for the risk of anemia.

This study examined the impact on anemia by modifying fatty acidprofiles through the diet in 20 MMP dolphins (“Group A dolphins”). Thedolphins lived in netted enclosures within San Diego Bay. The diets ofthe 20 Group A dolphins were modified from a 75% capelin (plus 25% mixof squid, herring or mackerel) baseline diet to a diet consisting of 25%capelin, 50% mullet, and 25% mix of squid, herring, or mackerel whilemaintaining the same kilocalories. On blood collection days, Group Adolphins were fed one-third of their daily diet in the morning aftertheir routine overnight fast and 2 h postprandial, in-water, and trainedblood samples were drawn (typically near 10:00 a.m.). An additional tenMMP dolphins (“Group B dolphins”) were maintained on the baselinecapelin diet throughout the study period. There were no differences inage, sex, or body weight when comparing the two groups (Table 2).

Capelin, the primary baseline fish type fed to MMP dolphins, had nodetectable C17:0 (<0.007 g/100 g). Mullet, the primary fish type fed inthe modified diet, had 67 mg/100 g of C17:0. For other fish, C17:0 wasmeasured as follows: herring=19 mg/100 g, and mackerel=22 mg/100 g.There was no detectable C17:0 in squid (See also Venn-Watson et al.(2016) Increased dietary intake of saturated fatty acid heptadecanoicacid (C17:0) associated with decreasing ferritin and alleviatedmetabolic syndrome in dolphins. PLOS ONE 10(7):e0132117).

Comparisons of daily fatty acid intake of the dolphins' baseline andmodified diets are provided (Table 3), including demonstrated increasedintake of odd chain saturated fatty acids C17:0 from a daily mean of 300to 1,100 mg (approximately a four-fold increase) and C15:0 from a dailymean of 1,300 to 4,500 mg (approximately a three-fold increase). Thevery long even chain saturated fatty acid, C24:0, increased from a dailymean intake of 0 to 300 mg. Given an approximate average body weight ofstudy dolphins of 159 kg, the modified diet provided an approximatedaily oral dose of C15:0 at 28 mg/kg body weight. The modifiedapproximate daily oral dose of C17:0 was 7 mg/kg body weight. Themodified approximate daily oral dose of C24:0 was 1.9 mg/kg body weight.When comparing the modified and baseline diets, there were no changes intotal fat intake, C18:0 or C22:0. Daily intake of the even chain fattyacids C14:0, C16:0, and C20:0, as well as total omega 3, totalpolyunsaturated, and total saturated fats decreased on the modifieddiet.

Two hour post-prandial samples were collected from Group A and Group Bdolphins at baseline (month 0) and at three time points following theswitch to the modified diet: months 1, 3, and 6. Dolphins were assessedfor changes in serum fatty acid concentrations (total and various forms)and indices of anemia, including hematocrit, packed cell volume, redblood cell count, hemoglobin, and red blood cell distribution.

Changes in serum fatty acid concentrations, including odd chainsaturated fatty acids and very long even chain saturated fatty acids, aswell as hematocrit, packed cell volume, red blood cell count, andhemoglobin were compared in study dolphins during months 1, 3, and 6 andcompared to month 0 using repeated measures ANOVA. Outcomes for markersof anemia for Group A and Group B dolphins are provided in Table 4. Allindicators of anemia improved in Group A dolphins by Month 1 and throughMonth 6. No changes were identified in Group B dolphins. It is apparentin FIG. 1. that individual Group A dolphins with low hematocrit hadresolving anemia while on the modified diet.

When the modified diet adding 50% mullet was fed to 20 Group A dolphinsover 6 months (increasing the average daily dietary C17:0 intake from300 to 1100 mg and C15:0 daily intake from 1,300 to 4,500 mg), totalserum odd chain saturated fatty acid concentrations increased by Month 1compared to Month 0 and maintained increased serum concentrationsthroughout the six months (Table 5). Specifically, at Month 1, serumtotal C15:0 concentrations increased to 94±33 uM, and serum total C17:0concentrations increased to 82±21 uM. Additionally, there were increasedconcentrations of multiple odd chain saturated fatty acid forms amongGroup A dolphins throughout the modified diet compared to Month 0,including raised C15:0 and C17:0 neutral fatty acids (for example,triacylglycerides) and raised C15:0 and C17:0 phospholipids (forexample, phosphatidylcholine and lysophosphatidylcholine) (Table 6).These changes in odd chain saturated fatty acid forms were not presentin Group B control dolphins. Thus, approximate daily oral dosing ofC15:0 at 28 mg/kg body weight or C17:0 at 7 mg/kg body weight may beused to achieve raised concentrations in these respective odd chainsaturated fatty acids.

When the modified diet adding 50% mullet was fed to 20 Group A dolphinsover 6 months, total serum concentrations of C26:0, a very long evenchain saturated fatty acid, increased by Month 1 compared to Month 0 andmaintained increased serum concentrations throughout the six months(Table 5). Additionally, there were increased concentrations of multipleadditional very long even chain saturated fatty acid forms among Group Adolphins throughout the modified diet compared to Month 0, includingraised serum C22:0 ceramide and hexosylceramide concentrations andraised C24:0 ceramide, dihydroceramide, and hexosylceramideconcentrations (Table 6). These changes in very long even chainsaturated fatty acid forms were not present in Group B control dolphins.The even chain saturated fatty acids, C16:0 and C18:0, either did notchange or decreased among Group A dolphins while on the modified diet.Thus, approximate daily oral dosing of C24:0 at 1.9 mg/kg body weightmay be used to achieve raised concentrations in very long even chainsaturated fatty acids.

Increased serum concentrations of odd chain saturated fatty acids inGroup A dolphins on the modified diet were independent, linearpredictors of improved red blood cell indices, or anemia (Table 7).Specifically, increased total serum concentrations of C15:0 and C17:0independently predicted higher hematocrit, higher packed cell volume,higher hemoglobin, and higher red blood cell counts. In addition,increases in concentrations of C15:0 cholesterol ester forms and C17:0phosphatidylcholine forms independently predicted improved red bloodcell indices. Plots 1 through 4 of FIG. 2 demonstrate the linearassociations between raised levels of these odd chain saturated fattyacid forms with raised hematocrit and raised hemoglobin. Thus, raisingserum concentrations of odd chain saturated fatty acids throughincreased daily oral intake of odd chain saturated fatty acidconcentrations can improve red blood cell indices and alleviate anemia.

Raised percentage of serum odd chain saturated fatty acids (of all fattyacids) in Group A dolphins on the modified diet was associated withimproved anemia (Table 7). Specifically, once dolphins reached a serumtotal C17:0 greater than 0.4%, only one dolphin remaining had anemia(hematocrit lower than 40%) (FIG. 3). Thus, achieving a total serumC17:0 value approximate to or greater than 0.4% of all fatty acidsprovided a threshold value and therapeutic target for improving anemia.

Increased serum concentrations of very long even chain saturated fattyacids in Group A dolphins on the modified diet were independent, linearpredictors of improved red blood cell indices (in other words, anemia)(Table 7). Specifically, increased concentrations of C20:0, C22:0, andC24:0 hexosylceramide forms independently predicted improved red bloodcell indices. Plots 5 through 7 of FIG. 4 demonstrate the linearassociations between raised levels of these very long even chainsaturated fatty acid forms with raised hematocrit. Thus, raising serumconcentrations of very long even chain saturated fatty acids can improvered blood cell indices and alleviate anemia.

Increased serum concentrations of odd chain saturated fatty acids thatwere independent predictors of improving anemia correlated withincreased serum concentrations of very long even chain saturated fattyacids that were independent predictors of improving anemia. Asdemonstrated in Plots 8-11 of FIG. 5, increasing serum concentrations ofC15:0 cholesterol ester forms had positive associations with increasingserum concentrations of C22:0 and C24:0 hexosylceramide forms.Increasing serum concentrations of C17:0 phosphatidylcholine forms hadpositives association with increasing serum concentrations of C22:0 andC24:0 hexosylceramide forms. Thus, increased serum concentrations of oddchain saturated fatty acid forms as a result of daily oral intake of oddchain saturated fatty acids are also associated with increases in serumconcentrations of targeted very long even chain fatty acid forms.

FIG. 6 summarizes results from the dolphin study, including that 1)increased oral intake of odd chain saturated fatty acids results inincreased serum concentrations of total odd chain saturated fatty acidsand a variety of odd chain saturated fatty acid forms, 2) increased oralintake of very long even chain saturated fatty acids results inincreased serum concentrations of very long even chain saturated fattyacid forms, 3) increased serum concentration of odd chain saturatedfatty acids due to increased odd chain saturated fatty acid oral intakeis associated with increased serum concentrations of very long evenchain saturated fatty acid forms, and 4) increased serum concentrationsof odd chain saturated fatty acids or very long even chain saturatedfatty acids independently predict linear improvement in red blood cellindices and improved anemia.

Sample Collection and Transport

Blood was collected into BD Vacutainer serum separator tubes (for serumfatty acid profiles) or an evacuated tube containing EDTA (K₃). Serumseparator tubes were centrifuged at 3000 rpm for 10 minutes within 30 to60 minutes of collection and chilled during processing until shipment.Serum was transferred to cryovials and stored at −80° C. until shipmenton dry ice via overnight courier to the reference laboratories.

Sample Analysis

Total serum fatty acid profiles were performed by the GeneticsLaboratories at the Kennedy Krieger Institute. Fatty acids were analyzedby capillary gas chromatography/mass spectrometry of pentaflourobenzylbromide fatty acid derivatives using an AT-Silar-100 column (Grace,Columbia, Md. 21044) as previously described. Each run was required topass clinical laboratory quality control before the data were released.CV % were typically under 10%. Percent fatty acids in serum was used asa sturdier index to help reduce potential variability in serum amongstudy dolphins.

Determination of serum fatty acid concentrations, including a variety offatty acid classes, were performed by Metabolon, Inc. (Durham, N.C.)using complex lipidomics. Lipids were extracted from samples usingdichloromethane and methanol in a modified Bligh-Dyer extraction in thepresence of internal standards with the lower, organic, phase being usedfor analysis. The extracts were concentrated under nitrogen andreconstituted in 0.25 mL of dichloromethane:methanol (50:50) containing10 mM ammonium acetate. The extracts were placed in vials forinfusion-MS analyses, performed on a SelexION equipped Sciex 5500 QTRAPusing both positive and negative mode electrospray. Each sample wassubjected to 2 analyses, with IMS-MS conditions optimized for lipidclasses monitored in each analysis. The 5500 QTRAP was operated in MRMmode to monitor the transitions for over 1,100 lipids from up to 14lipid classes. Individual lipid species were quantified based on theratio of signal intensity for target compounds to the signal intensityfor an assigned internal standard of known concentration. Lipid classconcentrations were calculated from the sum of all molecular specieswithin a class, and fatty acid compositions were determined bycalculating the proportion of individual fatty acids within each class.

Fish fatty acid profiles were performed by Covance Laboratories(Madison, Wis. 53703). Each of the following fish types was mixed withwater and homogenized for uniformity: capelin from Canada and Iceland(Mallotus villosus), herring (Clupea harengus), mackerel (Scomberjaponicus), squid (Loligo opalescens), and striped mullet (Mugilcephalus). The lipid was extracted, saponified with 0.5N methanolicsodium hydroxide, and methylated with 14% BF3-methanol. The resultingmethyl esters of the fatty acids were extracted with heptane. Aninternal standard was added prior to the lipid extraction. The methylesters of the fatty acids were analyzed by gas chromatography usingexternal standards for quantitation. These data were used to calculatetotal daily intake of each fatty acid for each study dolphin based uponrecorded diets eaten, by fish type, for their 1) pre-study baselinediet, and 2) in-study modified (Group A) or baseline (Group B) diets.

Red blood cell indices, including red blood cell count and hemoglobin,were performed by the Naval Medical Center San Diego. Betadine andalcohol swabs were used to clean the ventral aspect of the dolphin'sfluke using the appropriate aseptic technique. Blood was then drawn witha 21 g×¾ in. winged infusion set with a luer adapter and vacutainer hub.A BD vacutainer blood tube with 7.2 mg EDTA was then applied to the lueradapter and blood was evacuated using the vacuum from the tubes. Theblood was then shipped on ice packs to reference laboratories. A 4 mlEDTA vacutainer was shipped on an ice pack and analyzed by the NavalMedical Center of San Diego. The automated HCT, hemoglobin and red bloodcell count were analyzed using the Sysmex XE-5000 (Sysmex Canada Inc.,Mississauga, Ontario) per the manufacturer's protocol. For in-housepacked cell volume, whole blood was taken from a 4 mL BD Vacutainer tubewith 7.2 mg EDTA and used to fill a heparinized mylar wrapped 75 MMhematocrit tubes. Clay was packed into one end to prevent leakage duringcentrifugation. Once the hematocrit tube was sealed, it was placed intothe Thermo IEC MICRO-MB centrifuge (Thermo Fisher Scientific, Waltham,Ma. 02451). The blood sample was spun at 11,500 rpm for 5 minutes. Thehematocrit tube was then removed and placed into the micro-capillaryreader to determine results.

Statistical analyses were conducted using World Programming Systemsoftware (World Programming Ltd., Hampshire, United Kingdom).Significance was defined as a P value less than or equal to 0.05. Meandaily dietary intake of individual fatty acids were compared betweenpre-study and in-study diets for Group A and Group B dolphins usingWilcoxan rank sum tests. Red blood cell index values (hematocrit, packedcell volume, hemoglobin, and red blood cell counts) and serum fatty acidconcentrations (total and individual classes) from Month 1, 3, and 6were compared with Month 0 for Group A and Group B dolphins usingrepeated measures ANOVA (MIXED model). Fatty acids that had increasedserum concentrations on the modified diet, and not in controls, wereincluded in a stepwise regression model (for example, C17:0 total,triacylglyceride, phosphatidylcholine, lysophosphatidylcholine,phosphatidylethanolamine, and lysophosphatidylethanolamine forms wereincluded in one model) to determine which forms were independentpredictors of hematocrit, packed cell volume, hemoglobin, or red bloodcell counts. Fatty acids that were independent predictors of red bloodcell indices were then tested for linear associations with red bloodcell indices using simple linear regressions. Fatty acids that wereindependent redictors of red blood cell indices and had positive linearassociations with these indices (defined as a P value less than or equalto 0.05 and an R² greater than or equal to 0.1), were characterized asfatty acids that independently predicted improved anemia.

Table 1 provides comparisons of red blood cell indices, serum total oddchain fatty acids (% of total fatty acids) and serum total very longeven chain fatty acids (% of total fatty acids) between Navy MarineMammal Program (MMP) dolphins and wild Sarasota Bay dolphins.

TABLE 1 MMP Sarasota Dolphins Bay Dolphins Blood variable (n = 30) (n =19) P value Red blood cell index Packed cell volume (%)  38 ± 4  40 ± 60.008 Hemoglobin  13 ± 1  14 ± 2 0.001 Serum fatty acid (%) Odd chainsaturated fatty acids C15:0 0.3 ± 0.1 1.1 ± 0.4 <0.0001 C17:0 0.3 ± 0.11.3 ± 0.4 <0.0001 Very long even chain saturated fatty acids C20:0 0.9 ±0.2 1.5 ± 0.3 <0.0001 C22:0 0.2 ± 0.04 0.7 ± 0.2 <0.0001 C24:0 0.1 ± 0.00.5 ± 0.1 <0.0001

Table 2 provides comparisons of demographics between Group A dolphinsplaced on the modified fish diet and Group B dolphins maintained on thebaseline diet.

TABLE 2 Group A Group B control dolphins on dolphins modified dietremaining on P Demographic (n = 20) baseline diet (n-10) value Mean age(years)  22 ± 14  26 ± 10 0.41 Sex (no. and % female) 9/20 (45%) 4/10(40%) 0.79 Body weight (lbs) 389 ± 49 402 ± 45 0.33

Table 3 provides comparisons of total daily nutrient intake betweenbaseline and study diets for Group

TABLE 3 Case Dolphins Control Dolphins Baseline diet Modified Baselinediet Baseline diet Daily Nutrient Intake (pre-study) diet (pre-study)(during study) Total kilocalories 8,923 ± 2,411 9,139 ± 2,171 8,442 ±1,527 7,702 ± 2,897 Total pounds    16 ± 4    16 ± 4    15 ± 3    15 ± 3Total fat (g)   494 ± 133   460 ± 111*   468 ± 87   457 ± 89 Totalcarbohydrates (g)   25 ± 11   21 ± 5   20 ± 6   20 ± 6 Fatty acids (g)Odd chain saturated fatty acids C15:0  1.3 ± 0.4  4.5 ± 1.1*  1.2 ± 0.2 1.2 ± 0.3 C17:0  0.3 ± 0.1  1.1 ± 0.3*  0.3 ± 0.1  0.4 ± 0.2 Even chainsaturated fatty acids C16:0   58 ± 17   48 ± 14*   55 ± 9   53 ± 14C18:0  6.4 ± 1.9  7.6 ± 2.0  6.0 ± 0.9  6.1 ± 1.8 Very long even chainsaturated fatty acids C20:0  0.8 ± 0.2  0.4 ± 0.1*  0.7 ± 0.1  0.7 ± 0.2C22:0  0.1 ± 0.05  0.1 ± 0.04  0.1 ± 0.02  0.1 ± 0.1 C24:0  0  0.3 ±0.1*  0  0 Total omega 3   81 ± 23   60 ± 17*   77 ± 12   75 ± 15 Totalpolyunsaturated fats   84 ± 24   64 ± 18*   79 ± 13   77 ± 16 Totalsaturated fats   86 ± 25   73 ± 20*   81 ± 13   78 ± 19 Total fattyacids   338 ± 99   236 ± 66*   320 ± 53   305 ± 61 *P ≤ 0.05 compared tobaseline, pre-study diet

Table 4 provides comparisons of red blood cell index values among GroupA case dolphins on a modified diet and Group B control dolphin on abaseline diet.

TABLE 4 Red blood Case dolphins fed modified diet Control dolphinsmaintained on baseline diet cell (n = 20) (n = 10) index Month 0 Month 1Month 3 Month 6 Month 0 Month 1 Month 3 Month 6 Hematocrit 44 ± 5 46 ±3* 47 ± 2* 47 ± 2* 44 ± 3 44 ± 3 45 ± 3 44 ± 2 Packed cell 38 ± 4 41 ±2* 43 ± 2* 42 ± 2* 38 ± 3 39 ± 2 39 ± 2 39 ± 2 volume Red blood  3.0 ±0.3  3.2 ± 0.2*  3.4 ± 0.2*  3.4 ± 0.3*  3.0 ± 0.2  3.0 ± 0.2  3.2 ± 0.2 3.1 ± 0.2 cell count Hemoglobin 13 ± 1 14 ± 1* 15 ± 1* 15 ± 1* 13 ± 113 ± 1 14 ± 1 13 ± 1 *P ≤ 0.05

Table 5 provides comparisons of total serum fatty acid concentrationsamong Group A case dolphins on a modified diet and Group B controldolphin on a baseline diet.

TABLE 5 Total serum fatty acid concentration (uM) Case dolphins fedmodified diet Control dolphins maintained on baseline diet Fatty (n =20) (n = 10) acids Month 0 Month 1 Month 3 Month 6 Month 0 Month 1 Month3 Month 6 Odd chain saturated fatty acids C15:0 35 ± 9  94 ± 33*  111 ±29*  101 ± 29*  41 ± 19 45 ± 41  49 ± 19  38 ± 13 C17:0 51 ± 9  82 ± 21* 93 ± 23*  85 ± 15*  55 ± 17 53 ± 27 60 ± 9  50 ± 15 Even chainsaturated fatty acids C14:0 243 ± 63 195 ± 50 226 ± 46 206 ± 33 253 ± 90229 ± 114 294 ± 40 264 ± 92 C16:0 1445 ± 194 1372 ± 199 1505 ± 221 1450± 155 1521 ± 368 1411 ± 426  1639 ± 165 1484 ± 371 C18:0 1190 ± 229 1130± 188 1161 ± 194 1202 ± 140 1245 ± 348 1106 ± 379  1292 ± 184 1188 ± 344Very long even chain saturated fatty acids C20:0  94 ± 22  87 ± 17  98 ±17  96 ± 15  96 ± 27 84 ± 26 102 ± 14  91 ± 22 C22:0 23 ± 4 21 ± 3 23 ±3 24 ± 5 24 ± 5 21 ± 5  27 ± 5 23 ± 5 C24:0 18 ± 3 17 ± 3 18 ± 3 18 ± 319 ± 4 17 ± 4  20 ± 4 20 ± 4 C26:0  1.0 ± 0.1  0.8 ± 0.1*  0.9 ± 0.1* 0.9 ± 0.1*  1.1 ± 0.2 1.0 ± 0.2  1.1 ± 0.2  1.1 ± 0.2 *P ≤ 0.05

Table 6 provides comparisons of various serum fatty acid formconcentrations with significant (P≤0.05) changes from Month 0 in Group Acase dolphins on a modified diet. These changes were not present inGroup B control dolphin on a baseline diet (not shown).

TABLE 6 Serum concentrations (uM) with significant changes from Month 0in case dolphins fed modified diet (n = 20) Fatty acids Month 0 Month 1Month 3 Month 6 Odd chain saturated fatty acids C15:0 FFA  3.8 ± 1.3 4.2 ± 1.5  3.6 ± 0.8  3.8 ± 1.3 CE   22 ± 5   61 ± 19   66 ± 17   64 ±22 TAG  5.5 ± 1.9   11 ± 5   16 ± 10   12 ± 7 PC  5.4 ± 1.8   19 ± 8  22 ± 7   19 ± 7 LPC 0.91 ± 0.20  2.8 ± 0.9  2.9 ± 0.8  3.0 ± 1.1 C17:0CE   14 ± 4   18 ± 7   20 ± 8   21 ± 9 TAG  6.6 ± 2.1   11 ± 5   16 ± 10  11 ± 5 PC   15 ± 3   32 ± 9   34 ± 8   31 ± 7 LPC  1.8 ± 0.4  3.9 ±0.8  3.8 ± 0.8  3.9 ± 1.1 PE  1.3 ± 0.2  1.6 ± 0.6  1.9 ± 0.6  1.4 ± 0.2LPE 0.04 ± 0.01 0.09 ± 0.02 0.09 ± 0.02 0.09 ± 0.02 Even chain saturatedfatty acids C14:0 CE  126 ± 32   86 ± 21   93 ± 20   92 ± 25 C16:0 CE 273 ± 50  226 ± 41  244 ± 33  245 ± 48 CER 0.28 ± 0.04 0.24 ± 0.04 0.25± 0.05 0.23 ± 0.03 HCER 0.52 ± 0.12 0.41 ± 0.09 0.44 ± 0.09 0.43 ± 0.11SPM  126 ± 29  105 ± 20  112 ± 20  105 ± 23 C18:0 CER 0.59 ± 0.20 0.39 ±0.12 0.46 ± 0.18 0.40 ± 0.14 DCER 0.16 ± 0.05 0.10 ± 0.03 0.12 ± 0.050.11 ± 0.04 Very long even chain saturated fatty acids C20:0 FFA  6.6 ±2.5  4.4 ± 1.4  5.0 ± 2.3   17 ± 10 LPC 0.36 ± 0.08 0.27 ± 0.05 0.27 ±0.07 0.31 ± 0.09 CER 0.50 ± 0.10 0.39 ± 0.08 0.42 ± 0.08 0.41 ± 0.07HCER 0.04 ± 0.01 0.03 ± 0.01 0.03 ± 0.01 0.03 ± 0.01 C22:0 CE  8.5 ± 2.4 5.7 ± 1.5  5.9 ± 1.5  6.3 ± 2.1 CER 0.19 ± 0.04 0.23 ± 0.05 0.26 ± 0.070.25 ± 0.06 HCER 0.10 ± 0.02 0.12 ± 0.03 0.13 ± 0.03 0.13 ± 0.03 C24:0CER 0.38 ± 0.11 0.65 ± 0.17 0.75 ± 0.28 0.68 ± 0.19 DCER 0.07 ± 0.020.11 ± 0.03 0.13 ± 0.05 0.12 ± 0.03 HCER 0.16 ± 0.03 0.21 ± 0.05 0.22 ±0.05 0.22 ± 0.05 FFA = free fatty acid, CE = cholesterol ester, TAG =triacylglyceride, PC = phosphatidylcholine, LPC =lysophosphatidylcholine, PE = phosphatidylethanolamine, LPE =lysophosphatidylethanolamine, CER = ceramide, DCER = dihydroceramide,HCER = hexosylceramide

Table 7 provides serum fatty acids that were independent predictors ofimproving red blood cell indices in Group A dolphins while on a modifieddiet.

TABLE 7 P values for independent and linear serum fatty acid predictorsof improving red blood cell indices for Group A dolphins fed a modifieddiet (n = 20) Fatty acids Hematocrit Packed cell volume Hemoglobin Redblood cells Odd chain saturated fatty acids C15:0 Total 0.004 (+)<0.0001 (+) 0.002 (+) 0.0004 (+) FFA — — — — CE 0.0001 (+) <0.0001 (+)0.0001 (+) 0.0001 (+) TAG — — — — PC — — — — LPC — — — — C17:0 Total0.008 (+) 0.0006 (+) 0.006 (+) 0.004 (+) CE — — — — TAG — — — — PC0.0003 (+) 0.0001 (+) 0.0007 (+) 0.0003 (+) LPC — — — — PE — — — — LPE —— — — Even chain saturated fatty acids C14:0 CE — — — — C16:0 CE — — —0.003 (+) CER — — — — HCER — — — — SPM — — — — C18:0 CER — — — — DCER —— — — Very long even chain saturated fatty acids C20:0 FFA — — — — LPC —— — — CER — — — — HCER 0.003 (+) — 0.001 (+) <0.0001 (+) C22:0 CE — — —— CER — — — — HCER <0.0001 (+) 0.0003 (+) — <0.0001 (+) C24:0 CER — — —— DCER — — — — HCER <0.0001 (+) 0.0002 (+) 0.0001 (+) <0.0001 (+)

Odd chain saturated fatty acids and very long even chain saturated fattyacids are independent predictors among a full suite of red blood cellindices of anemia, including hematocrit, packed cell volume, hemoglobin,and red blood cell counts. When dolphins with anemia increased theirdietary intake of odd chain saturated fatty acids and very long evenchain fatty acids by changing fish types fed, hematocrit, packed cellvolume, hemoglobin, and red blood cell counts improved within 1 monthand maintained improved values throughout the six-month study. It isexpected that similar increases in odd chain saturated fatty acids andvery long even chain fatty acids will improve anemia in humans becausebottlenose dolphins (Tursiops truncatus) and humans are large-brained,long lived species that develop similar diseases, including conditionsassociated with aging, such as anemia. As such, dolphins have emerged asvaluable animal models relevant to human health.

Several parallels have been identified between dolphins and humans. Forexample, dolphins and humans are long-lived. The average lifespan ofdolphin is 20 years in the wild and 32 years at the MMP, with themaximum lifespan being approximately 60 years. Shared long lifespansbetween dolphins and humans are improving knowledge of chronic andaging-associated diseases in humans, including chronic anemia.Additionally, dolphins and humans have large brains. Among mammals,humans have the highest encephalization quotient (EQ=7.4), defined asthe actual versus expected brain size given a species' body size. Secondto humans is the bottlenose dolphin (EQ=5.3), higher than the chimpanzee(EQ=2.4) and much higher than the mouse (EQ=0.5).

Dolphins and humans have similar glucose transport systems, as well ascommon genetic adaptations associated with glucose metabolism. Adultdolphins have a high capacity for red blood cell glucose transport usingthe GLUT-1 transporter isoform; previous to this discovery, thiscapability was thought to be limited to primates. Common red blood cellglucose transport systems in cetaceans and primates are believed to bedue to high central nervous system glucose demands. Also, the dolphingenome has been partially sequenced by Baylor University, based upon adolphin at the U.S. Navy Marine Mammal Program. Dolphins have geneticevolutionary adaptations that are unique to long-lived, large brainedspecies, including humans and elephants. Further, dolphins and humanshave similar genes responsible for glucose metabolism (Office of NavalResearch funded study, unpublished). Accordingly, Dolphins areappropriate models for human metabolism.

Dolphins and Humans Develop Similar Diseases and Disease Complications

Similar to humans, common bottlenose dolphins (Tursiops truncatus) candevelop chronic diseases with advanced age, including chronicinflammation, dyslipidemia, and chronic anemia. Dolphins managed at theNavy Marine Mammal Program living in San Diego Bay, Calif., are awell-studied population with regard to metabolism, and this group haslower hematocrit and hemoglobin compared to a wild bottlenose dolphingroup living in Sarasota Bay, Fla. Importantly, the presence of case andreference populations of dolphins for anemia parallel similar humanpopulation comparisons.

Similar to humans, dolphins can develop a chronic condition involvinghigh ferritin (hyperferritinemia) and iron. This disease in humans anddolphins involves excessive iron deposition primarily in the liver'sKupffer cells, progression with age, and associations with elevatedlipids, insulin, and liver enzymes. This metabolic state in dolphins isassociated with neither mutations in the HFE gene. Contributor tohyperferritinemia in humans is chronic inflammation and associatedanemia of chronic disease.

Dolphins develop similar age-associated blood changes as aging humans.Specifically, absolute lymphocytes, serum globulins, and mean plateletvolume increase linearly with increasing old age (=aging from 30 up to50 years old). Mean white blood cells, neutrophils, serum globulins,erythrocyte sedimentation rates, serum cholesterol, and serumtriglycerides; and the prevalence of neutrophilic leukocytosis,hyperglobulinemia, and hypercholesterolemia, were more likely to behigher as geriatric dolphins got older. This study demonstrated thatolder dolphins have changes in hematological and serum chemistry valuessimilar to those found in older humans. As such, bottlenose dolphins canserve as a useful comparative model for aging in humans.

Dolphins and humans have a common evolutionary driver for sharedmetabolism. An increasingly accepted theory is that insulin resistancein humans evolved in ancestral primates during the last ice age. Duringthis time, our ancestors changed from a high carbohydrate, low proteindiet to a low carbohydrate, high protein diet. This change enabledgenetic selection of insulin resistance to help maintain blood sugarlevels needed for large brains. When humans returned to increasinglyhigh carbohydrate diets, however, insulin resistance became a pathologiccondition and led to type 2 diabetes. Approximately fifty-five millionyears ago, the dolphin was a terrestrial mammal that evolved to livecompletely in the marine environment. The closest terrestrial relativesof dolphins are artiodactyls, including cows, pigs, and camels. Most ofthese relatives are strict herbivores, and none are strict carnivores.As such, it may be assumed that the terrestrial ancestor of dolphins atea high carbohydrate, low protein diet. Similar to the evolutionary pathof humans during the ice age, dolphins changed to a high protein, lowcarbohydrate diet when they moved to the ocean. Because dolphins, too,have developed large brains with high demands for readily availableglucose, they may have also selected for glucose and lipid metabolismsimilar to humans.

For the above reasons, dolphins and humans share important common groundrelated to anatomy, physiology, and disease states that support thedolphin as an important and relevant animal model for human diseases,including anemia. The results cited herein for dolphins can also bebeneficial for humans.

The odd chain saturated fatty acids, C15:0 (pentadecanoic acid) andC17:0 (heptadecanoic acid), are present in ruminant whole dairy fat.Interestingly, despite consumer's movement away from high fat foods,whole fat dairy consumption in humans has been associated with multiplehealth benefits, including lower risks of insulin resistance syndrome,metabolic syndrome, and type 2 diabetes. To date, the mechanism of thebenefits of dairy products on human metabolism has not been determined.Based upon the results using the methods of the embodiments, it can beproposed that odd chain saturated fatty acids may be key players in thebenefits of dairy products to treating anemia in humans.

To take advantage of these benefits, odd chain saturated fatty acids andvery long even chain saturated fatty acids can be used in a supplement,food additive, food fortifier, beverage additive, beverage fortifier, orpharmaceutical in any form, including as a tablet, encapsulated pill,gelcap pill, liquid suspension, spray, and powder. Additionally,diagnostic tests and assays for odd chain saturated fatty acids and verylong even chain saturated fatty acids in human and animal samples(including blood (serum, plasma, and erythrocyte membranes), urine, andfeces) can be used to detect low odd chain saturated fatty acid and verylong even chain saturated fatty acid levels and to continually monitorthese fatty acid levels in patients. The use of odd chain saturatedfatty acids and very long even chain saturated fatty acids can stem andtreat conditions related to anemia and anemic conditions. Theseconditions include but are not limited to those related to anemicdisorders including, but not limited to hemolytic anemias (including butnot limited to thalassemias, hereditary spherocytosis, hereditaryelliptocytosis, glucose-6-phosphate dehydrogenase deficiency, pyruvatekinase deficiency, immune hemolytic anemia, alloimmune hemolytic anemia,drug-induced hemolytic anemia, mechanical hemolytic anemias, andparoxysmal nocturnal hemoglobinuria), anemia of chronic disease (whereinthe underlying condition can be, for example, autoimmune disorders (suchas, for example Crohn disease, systemic lupus erythematosus, rheumatoidarthritis and ulcerative colitis), neoplastic disorders including cancer(such as, for example lymphoma and Hodgkin disease), long-terminfections (such as, for example bacterial, viral, and fungalinfections), rheumatoid arthritis, ulcerative colitis, Hodgkin disease,metabolic syndrome, diabetes (for example, type 2 diabetes), and othercauses of chronic inflammation), anemia, aplastic anemias (including butnot limited to congenital hypoplastic anemia, Diamond-Blackfan anemiaand Fanconi anemia), iron deficiency anemia, anemias of abnormal RBCsize (including but not limited to megaloblastic anemia and microcyticanemia), vitamin deficiency anemias (including but not limited topernicious anemia) anemia of RBC mutation (including but not limited tothalassemia, sideroblastic anemia and sickle cell anemia). Theseegregious health effects can be treated or managed not only in dolphins,but because of the similarities in blood panels, they can be preventedin human mammals as well.

Referring now to FIG. 3, a scatter plot of C17:0 (as percent of serumfatty acids) versus hematocrit for the study population is shown. Asshown in FIG. 3, using a proposed therapeutic threshold of serum C17:0of 0.4 percent as percent of the total fatty acid in serums, canmaintain an adequately high hematocrit (greater than 40%).

Dolphins from MMP and Sarasota Bay live in the open ocean, and knowndietary intake was limited to fish fed to MMP dolphins. MMP dolphinslive in netted enclosures in San Diego Bay, and changing populations oflocal fish are readily available to eat. While MMP dolphins can eatlocal fish, however, observation of feeding behaviors by MMP's animalcare staff and maintained dolphin appetites for fed fish support thatthe majority of dietary fish are those that are fed by the MMP. ControlMMP (Group B) dolphins in the study did not have the same increases inred blood cell indices as Group A dolphins. The data suggest a directeffect for odd chain saturated fatty acids and very long even chainsaturated fatty acids on raising low red blood cell indices in thefeeding study involved fish with higher odd chain saturated fatty acidsand very long even chain saturated fatty acids. The potential impact (orcumulative impacts) of other non-fatty acid nutrients in the modifieddiet on red blood cell indices has not been determined. Identificationof 1) higher serum concentrations of odd chain saturated fatty acids andvery long even chain saturated fatty acids as independent predictors ofimproved red blood cell indices and anema, 2) demonstrated increaseddietary intake and serum concentrations of odd chain saturated fattyacids and very long even chain saturated fatty acids during the feedingstudy, and 3) coincident improvement in red blood cell indices andincreases in odd chain saturated fatty acids and very long even chainsaturated fatty acids by month 1, provide evidence that increasingdietary odd chain saturated fatty acids and very long even chainsaturated fatty acids contributed to raised red blood cell indices,which indicates that odd chain saturated fatty acids and very long evenchain saturated fatty acids can be used to treat anemia, as well asother associated or related conditions.

Odd chain saturated fatty acid and very long even chain saturated fattyacid deficiencies can be used to detect a risk of or cause for anemia.Dietary supplementation with odd chain saturated fatty acids and verylong even chain saturated fatty acids can help resolve anemia andassociated conditions.

Methods and compositions related to or applicable to anemia or relatedconditions are discussed in the following references, which areincorporated by reference herein in their entirety: Fave, G. (2004)Physiochemical properties of lipids: new strategies to manage fatty acidbioavailability. Cell Bol Biol 50:815-831. Ramirez, M. (2001) Absorptionand distribution of dietary fatty acids from different sources. EarlyHum Develop 65:S95-S101; Yin, G. (2015) Concurrent Epstein-Barr virusassociated NK/T cell lymphoma after immunosuppressive therapy foraplastic anemia: report of a case and review of literature.International Journal of Clinical and Experimental Pathology, 8(6),7588-7593; Barcellini, W. (2015) Clinical Applications of HemolyticMarkers in the Differential Diagnosis and Management of HemolyticAnemia. Disease Markers, 2015, 635670; Carnaschella, C. (2015)Iron-deficiency Anemia, N. Engl. J Med. 372:19; Oberley, M. J. and Yang,D. T. (2013), Laboratory testing for cobalamin deficiency inmegaloblastic anemia. Am. J. Hematol., 88: 522-526; Hebbel, R. P.,Vercellotti, G. M., & Nath, K. A. (2009). A Systems BiologyConsideration of the Vasculopathy of Sickle Cell Anemia: The Need forMulti-Modality Chemo-Prophylaxis. Cardiovascular & HematologicalDisorders Drug Targets, 9(4), 271-292; Fujiwara, T., & Harigae, H.(2015). Biology of Heme in Mammalian Erythroid Cells and RelatedDisorders. BioMed Research International, 2015, 278536; Craik J. (1998)GLUT-1 mediation of rapid glucose transport in dolphin (Tursiopstruncatus) red blood cells. Am J Physiol 274:R112-R9; McGowen M. (2012)Dolphin genome provides evidence for adaptive evolution of nervoussystem genes and a molecular rate slowdown. Royal Society Proc B279:3643-51; Jenkins B. (2015) A review of odd-chain fatty acidmetabolism and the role of pentadecanoic acid (C15:0) and heptadecanoicacid (C17:0) in health and disease. Molecules 20:2425-44; Mansson H L(2008) Fatty acids in bovine milk fat. Food Nutr Res 52:4; Luzia L A.(2013) The influence of season on the lipid profiles of fivecommercially important species of Brazilian fish. Food Chem 83:93-97;Benatar J R. (2014) The effects of changing dairy intake on trans andsaturated fatty acid levels—results from a randomized controlled study.Nutr J 13:32; Abdullah M M. (2015) Recommended dairy product intakemodulates circulating fatty acid profile in healthy adults: amulti-centre cross-over study. Br J Nutr 113:435-44; Forouhi N. (2014)Differences in the prospective association between individual plasmaphospholipid saturated fatty acids and incident type 2 diabetes: theEPIC-InterAct case-cohort study. Lancet Diabetes Endocrinol 2:810-8;Patel P. (2010) Fatty acids measured in plasma and erythrocyte-membranephospholipids and derived by food-frequency questionnaire and the riskof new-onset type 2 diabetes: a pilot study in the European ProspectiveInvestigation into Cancer and Nutrition (EPIC)-Norfolk cohort. Am J ClinNutrition 92:1214-22; Krachler B. (2008) Fatty acid profile of theerythrocyte membrane preceding development of Type 2 diabetes mellitus.Nutrition, metabolism, and cardiovascular diseases. NMCD 18:503-10;Maruyama C. (2008) Differences in serum phospholipid fatty acidcompositions and estimated desaturase activities between Japanese menwith and without metabolic syndrome. J Atherscler Thromb 15:306-313;Choi H. (2005) Dairy consumption and risk of type 2 diabetes mellitus inmen: a prospective study. JAMA Internal Med 165:997-1003; Kratz M.(2014) Dairy fat intake is associated with glucose tolerance, hepaticand systemic insulin sensitivity, and liver fat but not beta-cellfunction in humans. The American journal of clinical nutrition.99:1385-96; Barros N B. (1998) Prey and feeding patterns of residentbottlenose dolphins (Tursiops truncatus) in Sarasota Bay, Fla. J Mammal79:1045-1059; Berens-McCabe E. (2010) Prey selection in a residentcommon bottlenose dolphin (Tursiops truncatus) community in SarasotaBay, Fla. Marine Biol 157:931-942; Lagerstedt S A. (2001) Quantitativedetermination of plasma C8-C26 total fatty acids for the biochemicaldiagnosis of nutritional and metabolic disorders. Mol Gen Metabol73:38-45; Benatar J R. (2014) The effects of changing dairy intake ontrans and saturated fatty acid levels-results from a randomizedcontrolled study. Nutr J 13:32. doi: 10.1186/1475-2891-13-32PMID:24708591 Venn-Watson S. (2015) Annual survival, mortality, andlongevity of bottlenose dolphins (Tursiops truncatus) at the U.S. NavyMarine Mammal Program, 2004-2013. J Am Vet Med 246:893-898; Venn-WatsonS. (2008) Clinical relevance of elevated transaminases in a bottlenosedolphin (Tursiops truncatus) population. J Wildlf Dis 44:318-330; Hall,A. J. (2007). Annual, seasonal and individual variation in hematologyand clinical blood chemistry profiles in bottlenose dolphins (Tursiopstruncatus) from Sarasota Bay, Fla. Comp Biochem Physiol A Mol IntegrPhysiol 148, 266-277. doi: 10.1016/j.cbpa.2007.04.017;

The above description presents the best mode contemplated for carryingout the present invention, and of the manner and process of making andusing it, in such full, clear, concise, and exact terms as to enable anyperson skilled in the art to which it pertains to make and use thisinvention. This invention is, however, susceptible to modifications andalternate constructions from that discussed above that are fullyequivalent. Consequently, this invention is not limited to theparticular embodiments disclosed. On the contrary, this invention coversall modifications and alternate constructions coming within the spiritand scope of the invention as generally expressed by the followingclaims, which particularly point out and distinctly claim the subjectmatter of the invention. While the disclosure has been illustrated anddescribed in detail in the drawings and foregoing description, suchillustration and description are to be considered illustrative orexemplary and not restrictive.

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Unless otherwise defined, all terms (including technical and scientificterms) are to be given their ordinary and customary meaning to a personof ordinary skill in the art, and are not to be limited to a special orcustomized meaning unless expressly so defined herein. It should benoted that the use of particular terminology when describing certainfeatures or aspects of the disclosure should not be taken to imply thatthe terminology is being re-defined herein to be restricted to includeany specific characteristics of the features or aspects of thedisclosure with which that terminology is associated. Terms and phrasesused in this application, and variations thereof, especially in theappended claims, unless otherwise expressly stated, should be construedas open ended as opposed to limiting. As examples of the foregoing, theterm ‘including’ should be read to mean ‘including, without limitation,’‘including but not limited to,’ or the like; the term ‘comprising’ asused herein is synonymous with ‘including,’ ‘containing,’ or‘characterized by,’ and is inclusive or open-ended and does not excludeadditional, unrecited elements or method steps; the term ‘having’ shouldbe interpreted as ‘having at least;’ the term ‘includes’ should beinterpreted as ‘includes but is not limited to;’ the term ‘example’ isused to provide exemplary instances of the item in discussion, not anexhaustive or limiting list thereof, adjectives such as ‘known’,‘normal’, ‘standard’, and terms of similar meaning should not beconstrued as limiting the item described to a given time period or to anitem available as of a given time, but instead should be read toencompass known, normal, or standard technologies that may be availableor known now or at any time in the future; and use of terms like‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction of the invention, but instead as merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment of the invention. Likewise, a group of itemslinked with the conjunction ‘and’ should not be read as requiring thateach and every one of those items be present in the grouping, but rathershould be read as ‘and/or’ unless expressly stated otherwise. Similarly,a group of items linked with the conjunction ‘or’ should not be read asrequiring mutual exclusivity among that group, but rather should be readas ‘and/or’ unless expressly stated otherwise.

Where a range of values is provided, it is understood that the upper andlower limit, and each intervening value between the upper and lowerlimit of the range is encompassed within the embodiments.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. The indefinite article ‘a’ or ‘an’ does not exclude aplurality. A single processor or other unit may fulfill the functions ofseveral items recited in the claims. The mere fact that certain measuresare recited in mutually different dependent claims does not indicatethat a combination of these measures cannot be used to advantage. Anyreference signs in the claims should not be construed as limiting thescope.

It will be further understood by those within the art that if a specificnumber of an introduced claim recitation is intended, such an intentwill be explicitly recited in the claim, and in the absence of suchrecitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases ‘at least one’ and “one or more’ to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles ‘a’ or ‘an’ limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases‘one or more’ or ‘at least one’ and indefinite articles such as ‘a’ or‘an’ (e.g., ‘a’ and/or ‘an’ should typically be interpreted to mean ‘atleast one’ or ‘one or more’); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of ‘two recitations,’ without othermodifiers, typically means at least two recitations, or two or morerecitations). Furthermore, in those instances where a conventionanalogous to ‘at least one of A, B, and C, etc.’ is used, in generalsuch a construction is intended in the sense one having skill in the artwould understand the convention (e.g., ‘a system having at least one ofA, B, and C’ would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). In those instances where aconvention analogous to ‘at least one of A, B, or C, etc.’ is used, ingeneral such a construction is intended in the sense one having skill inthe art would understand the convention (e.g., ‘a system having at leastone of A, B, or C’ would include but not be limited to systems that haveA alone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase ‘A or B’ will be understood toinclude the possibilities of ‘A’ or ‘B’ or ‘A and B.’

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term ‘about.’ Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Furthermore, although the foregoing has been described in some detail byway of illustrations and examples for purposes of clarity andunderstanding, it is apparent to those skilled in the art that certainchanges and modifications may be practiced. Therefore, the descriptionand examples should not be construed as limiting the scope of theinvention to the specific embodiments and examples described herein, butrather to also cover all modification and alternatives coming with thetrue scope and spirit of the invention.

What is claimed is:
 1. A method comprising: using pentadecanoic acid ina supplement to human diet at a level sufficient to treat symptoms ofanemia, wherein said supplement is selected from the group consisting offood additives, food fortifiers, beverage additives, beveragefortifiers, or pharmaceuticals, and is administered to a subject at adaily dose comprising an approximate minimum of 3 mg per lb (or 6 mg perkg) of body weight.
 2. A method comprising: administering, to a patient,pentadecanoic acid at a daily dose comprising an approximate minimum of3 mg per lb (or 6 mg per kg) of the patient's body weight, wherein thepentadecanoic acid is administered in a form selected from the groupconsisting of a supplement, a food, a food additive, a food fortifier, abeverage additive, a beverage fortifier, and a pharmaceutical.
 3. Themethod of claim 2, wherein the daily dose is at a level sufficient totreat symptoms of anemia.
 4. The method of claim 2, wherein the dailydose is at a level sufficient to support production, formation andmaintenance of healthy red blood cells.
 5. The method of claim 2,wherein the daily dose is at a level sufficient to support healthy bloodcell replication.
 6. The method of claim 2, wherein the daily dose is ata level sufficient as a hemoglobin blood enhancer.
 7. The method ofclaim 2, wherein the daily dose is at a level sufficient to reduce therisk of anemia.
 8. The method of claim 2, wherein the pharmaceutical isin a form selected from the group consisting of a tablet, anencapsulated pill, a gelcap pill, a liquid suspension, a spray, and apowder.
 9. The method of claim 2, wherein the patient is a mammal. 10.The method of claim 9, wherein the mammal is a human.